Compositions and their use for removing cholesterol

ABSTRACT

The invention is directed to compositions that function to remove cholesterol from a mammal suffering from an elevated cholesterol level. The composition includes a polysaccharide having attached thereto at least one cyclic oligosaccharide. In a particular embodiment, the foregoing composition further includes at least one cell-targeting agent. The invention is also directed to methods that utilize these compositions for removing or reducing cholesterol and other lipids in a mammal suffering from an elevated level of cholesterol and/or other lipid.

CROSS REFERENCE TO RELATED APPLICATION

This application claims the benefit of priority from U.S. ProvisionalApplication No. 61/181,996, filed on May 28, 2009.

GOVERNMENT SUPPORT

This invention was made with government support under GM079238 awardedby the National Institutes of Health. The government has certain rightsin the invention.

FIELD OF THE INVENTION

The present invention relates, generally, to compositions useful forremoval or reduction of cholesterol in living tissue, as well as methodsfor their use in treating a mammal suffering from an elevated level ofcholesterol as caused by environmental or genetic factors.

BACKGROUND OF THE INVENTION

Several conditions and disorders are characterized by an elevated levelof cholesterol in tissues of the body. New, safer, and more effectivemethods for reducing cholesterol levels in individuals with elevatedlevels of cholesterol (e.g., hypercholesterolemia and atheroschlerosis)are continually sought. Furthermore, there are continuing efforts totreat individuals suffering from inherited disorders that exhibit as aprimary symptom an accumulation of lipids in tissues and cells of thebody. These inherited disorders typically belong to the class of lipidstorage disorders (LSDs), of which a notable example is Niemann-Pick(NP) disease. The etiology of LSDs is generally a malfunction of thedegradative function of the lysosome, and more specifically, a result ofan insufficient production of, or diminished function of, a metabolizingenzyme of the lysosome.

SUMMARY OF THE INVENTION

The invention is directed, in a first aspect, to compositions thatfunction to remove cholesterol from a mammal suffering from an elevatedcholesterol level. In a particular embodiment, the composition includesa polysaccharide having attached thereto at least one cyclicoligosaccharide. In a further embodiment, the foregoing compositionfurther includes at least one cell-targeting agent. Alternatively, thecomposition includes an active portion containing a polysaccharidehaving attached thereto at least one cyclic oligosaccharide, wherein atleast one cell-targeting agent is attached to the active portion.

In a second aspect, the invention is directed to a pharmaceuticalcomposition of any of the above-described compositions. Thepharmaceutical composition includes the above-described composition in apharmaceutically acceptable vehicle.

In a third aspect, the invention is directed to methods for removingcholesterol from mammalian tissue, or treating a mammal suffering froman elevated cholesterol level or suffering from a lysosomal storagedisorder, by administering to the mammal any of the pharmaceuticalcompositions described above.

In a fourth aspect, the invention is directed to methods for removingcholesterol from mammalian tissue, or treating a mammal suffering froman elevated cholesterol level or suffering from a lysosomal storagedisorder, by administering to the mammal at least one histonedeacetylase (HDAC) inhibitor.

In a fifth aspect, the invention is directed to methods for removingcholesterol from mammalian tissue, or treating a mammal suffering froman elevated cholesterol level or suffering from a lysosomal storagedisorder, by administering to the mammal at least one sphingomyelinaseenzyme.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1. Graph showing the concentration and time dependence of theremoval of cholesterol from LSOs of human NPC1 cells incubated with theindicated concentrations of MβCD in complete medium for 1-4 days.Similar results were obtained with NPC2 cells.

FIG. 2. Graph showing concentration of cholesterol from LSOs of NPC1cells incubated with 0.33 mM MβCD for one hour under various conditions(i.e., no FBS, 10% FBS, and cholesterol-loaded MβCD (i.e.,“MbCD/cholest.”), and these with either no chase or a 24 hour chase). Atthe end of the incubation, cells were rinsed thoroughly, and returned tocomplete medium for 24 hours. Although a one-hour incubation withcholesterol-loaded MβCD increased the cholesterol in LSOs, by 24 hoursthe LSO cholesterol in these cells was greatly reduced, even withnegligible amounts of extracellular MβCD. Similar results were obtainedwith NPC2 cells.

FIG. 3. Graph comparing the cholesterol-lowering ability of MβCD and aMβCD-dextran composition of the instant invention. Human NPC1 cells wereincubated with MβCD () or Dextran-PEG-MbCD (O) for 39 hours. Thepolymers had about two MβCDs per polymer, and the concentration is theMβCD concentration. Since two MβCD molecules are required to solubilizea cholesterol, it is likely that higher degrees of substitution would bemore effective, but even these first polymers show a significant effect.

FIG. 4. Micrographs showing the effect of CDs on cholesterolaccumulation in NPC1- and NPC2-deficient cells. (A) Background andshading corrected images of untreated (a and b), MβCD-treated (c and d),or HPβCD-treated (e and f) GM03123 NPC1 cells (a, c, and e) or GM18455NPC2 (b, d, and f) mutant cells. NPC mutant cells were treated with 300μMCD for 1 day. (B) Untreated, apparently normal GM05659 cells, shownfor comparison. (Scale bars, 100 μm.)

FIGS. 5A-D. Charts showing quantification of CD effects on cholesterolaccumulation in NPC1 and NPC2 mutant cells. The filipin fluorescence inLSOs was measured in NPC1 (A and B) and NPC2 (C and D) mutant cellstreated with either MβCD (A and C) or HPβCD (B and D) for 1-4 days. Dataare presented as percentage of the average value for untreated controlsin two independent experiments±SEM (n=6 for treated samples, n=40 forcontrol, where n is total number of wells per condition used forquantification).

FIG. 6. Chart comparing cholesterol accumulation as measured by the LSOassay in NPC1-defective, NPC2-defective, and apparently normal humanfibroblasts. Cells were plated in 384-well assay plates at a density toproduce ≈80-90% confluency after 4 days. At the end of the experiment,cells were fixed with 1% PFA and stained with 50 μg/mL filipin. Imageswere acquired using an ImageXpress MICRO imaging system with a 10×objective. Quantification of filipin labeling of the LSOs was performedas described previously (Pipalia N H, Huang A, Ralph H, Rujoi M,Maxfield F R (2006) Automated microscopy screening for compounds thatpartially revert cholesterol accumulation in Niemann-Pick C cells. J.Lipid Res. 47:284-301). Data represent averages±SEM of onerepresentative experiment (11≦n≦16, where n is total number of wells percondition used for quantification).

FIGS. 7 A-D. Charts showing GC/MS measurement of cyclodextrin effects ontotal free cholesterol levels in NPC1-deficient, NPC2-deficient,apparently normal, and U18666A-treated normal cells. Cells were platedin six-well plates at a density to produce ≈40-90% confluency byexperiment completion. Cells were treated with 0.9 mM MβCD or HPβCD for1 day in growth medium. At the end of the experiment, cellular lipidswere extracted and analyzed by GC/MS. Data represent averages±SEM of twoindependent experiments normalized to control average value for eachexperiment. Control average values were as follows: 0.070±0.011 pmolfree cholesterol per μg cell protein for NPC1 mutant cells (A),0.133±0.005 pmol/μg for NPC2 mutant cells (B), 0.044±0.003 pmol/μg fornormal cells (C), and 0.074±0.006 pmol/μg for normal cells treated with1 μM U18666A (D). *P<0.01 vs. control (6≦n≦9, where n is total number ofwells per condition used for quantification).

FIGS. 8A-D. Charts showing persistence of CD effects after treatment.NPC1 (A and B) and NPC2 (C and D) treated with various concentrations ofMβCD (A and C) and HPβCD (B and D) for 1 day and then fixed (0 daysafter 24 h treatment), or rinsed extensively, and allowed to grow innormal growth medium for up to three additional days after CD removal.Quantification of LSO filipin fluorescence power is shown. Data arepresented as percentage of the average value for untreated controls intwo independent experiments±SEM (n=6 for treated samples, n=24 forcontrol, where n is total number of wells per condition used forquantification).

FIGS. 9A, B. Filipin micrographs showing acute cyclodextrin effects oncholesterol accumulation in NPC1- and NPC2-deficient cells. Backgroundand shading corrected images of untreated control (panels a-c), 0 hafter treatment (panels d-f), or 24 h after treatment (panels g-i) forNPC1 (A) or NPC2 (B) mutant cells were obtained as in FIG. 6. Cells weretreated either with 333 μM M3CD dissolved in MEM with no serum (panelsa, d, and g), in MEM with 10% FBS (panels b, e, and h), or with 333 μMMβCD loaded with cholesterol (panels c, f, and i). (Scale bars, 100 μm.)

FIGS. 10A-D. Short-term MβCD treatment. NPC1 (A and B) and NPC2 (C and.D) mutant cells were treated for 1 hour with 333 μM MβCD in growth mediawithout serum, 333 μM MβCD in media plus 10% FBS, or 333 μM MβCD loadedwith cholesterol (5:1 (MβCD/cholesterol) ratio). Cells were either fixedimmediately or rinsed extensively and returned to growth medium for 24hours. Quantification of LSO filipin fluorescence power is shown (A andC). Numeric data represent averages±SEM of three independent experimentsnormalized to control (untreated) average value for each experiment.*P<0.0001 vs. control (n=12, where n is total number of wells used forquantification). Filipin images of NPC1 (B) or NPC2 (D) mutant cellstreated with MβCD/cholesterol are shown. (Scale bars, 100 μm.)

FIGS. 11A, B. MβCD-dextran localization and effects on LSO filipin inNPC1-defective cells. (A) Cells were treated for 2 days with 50 μM MβCDor 50 μM MβCD conjugated to dextran fixed with paraformaldehyde,stained, and imaged. Cholesterol accumulation in LSOs was measured byquantifying filipin fluorescence. Data represent averages±SEM of onerepresentative experiment (n=8, where n is total number of wells percondition used for quantification). (B) Cells were incubated withAlexaFluor546-MβCD-dextran for 15 hours followed by a 3-hour chase ingrowth medium. The cells were fixed and imaged by epifluorescencemicroscopy. The MβCD concentration was 300 μM, with ≈2 MβCD per 72-kDadextran chain. (Scale bars, 11 μm.)

FIGS. 12A, B. Charts showing cholesterol esterification by ACAT.Cyclodextrin effects on cholesterol esterification in NPC1 (A) and NPC2(B) mutant cells. Cells were treated for 1 hour with 3 mM MβCD or HPβCDand then allowed to recover in growth medium for 16 hours. Cells werethen incubated with 3 μM4,4-difluoro-4-bora-3a,4a-diaza-s-indacene-dodecanoic acid (BODIPY-C12)for 6 hours. The ratio of cholesteryl-BODIPY-C12 ester (CE)/BODIPY-C12acid (FA) after TLC separation was measured and normalized to theaverage control value. ACAT inhibitor S58-035 was added, whereindicated, at 10 μg/mL. For NPC1-defective cells, the average ratio was0.110±0.007, which corresponded to 3.73±0.22 Pmol CE formed per μg cellprotein during 6 hours. Corresponding values for NPC2-defective cellswere 0.089±0.003 and 2.50±0.24 fmol/μg. Data represent averages±SEM oftwo to three independent experiments. *P<0.0001 vs. control (6≦n≦16,where n is total number of wells per condition used for quantification).

FIGS. 13A, B. Charts showing LSO filipin values for the ACAT assay. LSOfilipin levels were measured immediately (no chase) or after 24-hourchase after a 1-hour 3-mM MβCD treatment in NPC1 (A) and NPC2 (B) mutantcells. Data represent averages±SEM of two independent experiments (n=8for treated samples, n=72 for untreated control, where n is total numberof wells per condition used for quantification).

FIGS. 14A-D. Cyclodextrin effects on cholesterol accumulation inU18666A-treated apparently normal human fibroblasts. Cells were platedin 384-well assay plates at a density to produce ≈80-90% confluency byexperiment completion and treated with U18666A at indicatedconcentrations for 24 hour before addition of various concentrations ofeither MβCD or HPβCD for another 24 hours, during which indicatedU18666A levels were maintained. At the end of the experiment, cells werefixed with 1% PFA and stained with 50 μg/mL filipin. Images wereacquired using an ImageXpressMICRO imaging system with a 10× objective.LSO filipin quantification was performed as described previously (N. H.Pipalia, et al., Ibid.). (A) Cholesterol accumulation, as measured bythe LSO filipin assay, at different concentrations of U18666A. MβCD (B)and HPβCD (C) LSO filipin dose-response curves at three differentU18666A concentrations. (D) MβCD and HPβCD EC50 (from Table 2)dependence on U18666A concentration. Data represent averages±SEM of twoindependent experiments (n=12 for control and n=6 for treated, where nis total number of wells per condition used for quantification).

FIGS. 15A-C. CD effects on BMP accumulation in NPC mutant cells. BMPlevels were quantified using fluorescence microscopy from imagesobtained with a 20× objective of cells stained with anti-BMP antibodyand AlexaFluor488-conjugated secondary antibody in NPC1 (A), NPC2 (B)mutant, and 1 μM U18666A-treated normal (C) cells. Cells were incubatedwith 0.9 mM CD for ≈1 day. Data represent averages±SEM of twoindependent experiments normalized to control (untreated) average valuefor each experiment. *P<0.0001 vs. control (n=16 for treated samples,n≈64 for control, where n is total number of wells per condition usedfor quantification).

FIG. 16. Comparison of BMP accumulation in NPC1-defective,NPC2-defective, normal, and U18666A-treated normal fibroblasts. BMPlevels were quantified using fluorescence microscopy from 20×magnification images obtained with anti-BMP antibody (clone 6C4) andAlexaFluor488-conjugated goat anti-mouse secondary antibody. Datarepresent averages±SEM of one representative experiment (n=32, where nis total number of wells per condition used for quantification).

FIG. 17. Chemical structures of various HDAC inhibitor compounds(bufexamac, C1-994, pyroxamide, SAHA, trichostatin A, LBH-589).

FIGS. 18A, B. Graphs showing the dose dependent effect of HDACinhibitors on cholesterol accumulation in human NPC1 fibroblasts. Cellswere treated in complete medium for two days (48 hours). The dashedhorizontal line is 3SD below the mean for untreated cells. (A) resultsfor NPC1 human fibroblast GM18453, (B) results for NPC2 human fibroblastGM 18445.

FIG. 19. Plots showing the dose and time dependence for each of thestudied HDACi compounds as a function of time on NPC1 fibroblastGM03123.

FIG. 20. Plots showing the effect of various HDACi compounds on cellproliferation. The effect was monitored at 4 h, 24 h, 48 h and 72 h posttreatment by counting the number of nuclei. Each data point in a plot isrepresentative of a total of 32 images from two independent experiments.Error bar=SE.

FIG. 21. Plot showing a filipin assay after HDAC silencing in GM03123cells by electroporation. The plot is representative of threeindependent experiments for HDAC 6 and two independent experiments forHDAC7A.

FIGS. 22A-F. Decrease in cholesterol accumulation in LSOs by restorationof acid SMase activity in CT60 cells. A) Acid SMase activity plot: CT60cells were transfected with empty vector (VEC) or with vector containingeither the WT or the C629S SMPD1 cDNA constructs. Two days later,extracts of these cells and non-transfected 25RA. and CT60 cells wereassayed for acid SMase activity. The activity levels in CT60-WT andCT60-C629S cells were significantly different from those innon-transfected CT60 cells and CT60-VEC cells (p<0.0001). B)Micrographs: the five cell groups described in (A) were fixed andstained with filipin. The images are displayed with the same gray scalerange. Scale bar, 20 μm. C) LSO filipin intensity plot: quantificationof filipin fluorescence in the LSOs, and D) in whole cells. Each bar inthe data quantification represents the average of 30 images from twoindependent experiments±SEM. The CT60-WT and CT60-C629S values weresignificantly different from the CT60 and. CT60-VEC values (p<0.0001).NB: The 25RA and. CT60 cells used in this experiment are the same onesused in FIG. 26, i.e. they express the hTfR. However, they have the samelevel of cholesterol accumulation in LSOs and the same response to acidSMase restoration as cells not expressing the TfR (data not shown). E)Plot of relative cellular cholesterol mass: free cholesterol levels ineach of the five cell groups described in (A) were assayed by gaschromatography. Each bar represents an average of four samples from twoindependent experiments and is normalized to CT60-VEC value, which was45.7±1.7 μg cholesterol/mg cell protein. The CT60-WT and CT60-C629Svalues were significantly different from the CT60 and CT60-VEC values(p<0.0001). F) Monolayers of the five groups of cells described abovewere incubated for 2 hours with 5 μg/mL LDL reconstituted with[¹⁴C]cholesteryl ester (CE). Lipid extracts of the cells were thenfractionated by thin-layer chromatography, and the areas of the platecorresponding to cholesterol and CE, which accounted for all of theradioactivity, were scraped and counted for [¹⁴C]cpm. [¹⁴C]cholesterolrepresents hydrolyzed LDL-CE in the cells, and [¹⁴C]CE represents eitherunhydrolyzed LDL-CE or hydrolyzed LDL-cholesterol that was re-esterifiedin the cells to CE. The data shown are derived from the totalLDL-derived cholesterol in the cells (free cholesterol+CE) and are meanof 5 values±SEM. The values for cellular-free cholesterol derived fromLDL were similar among all the five cell types: 1.92, 1.88, 1.93, 2.05,1.72 pmol/μg cell protein, respectively. None of the differences intotal or free LDL-derived cellular cholesterol among the five groups ofcells reached statistical significance.

FIGS. 23A, B. Partial correction of efflux of LDL-derived[³H]cholesterol from CT60 cells by restoration of acid SMase activity.A) Monolayers of 25RA, CT60, CT60-VEC, CT60-WT and CT60-C629S wereincubated for 4 h in serum-free medium containing 10 μg/mL[³H]CE-labeled LDL. The cells were then rinsed and incubated with freshmedium containing 50 μg/mL HDL3 for the indicated times. Tritiumradioactivity in the media and cells was measured to calculate thepercent [³H]cholesterol in the medium. The values for CT60-WT andCT60-C629S cells were significantly different from the other threevalues at 24 h (p<0.005). B) The bottom graph shows acid SMase activityin the five groups of cells at 0, 8 and 24 hours after incubation inconditions nearly identical to those in (A), except that cells wereincubated with unlabeled LDL. The values for CT60-WT and CT60-C629Scells were significantly different from that of CT60 and CT60-VEC(p<0.005).

FIGS. 24A, B. Decrease in BMP accumulation in CT60 cells by restorationof acid SMase activity. A) Anti-BMP immunofluorescence in 25RA, CT60,CT60-VEC, CT60-WT and CT60-C629S cells. Scale bar, 20 μm. B)Quantitation of anti-BMP immunofluorescence intensity. Each bar in thedata quantification represents the average of 20 images from twoindependent experiments±SEM. The values for CT60-WT and CT60-C629S cellswere significantly different from that of CT60 and CT60-VEC cells(p<0.01). NB: As in FIG. 1, the cells used here express the hTfR, butthey have the same level of BMP accumulation in LSOs and the sameresponse to acid SMase restoration as the cells not expressing the TfR(data not shown).

FIG. 25. Plot showing that recycling of the transferrin receptor in CT60cells is improved by restoration of acid SMase activity. The effluxkinetics of [¹²⁵I]-transferrin was measured in 25RA, CT60, CT60VEC,CT60-WT and CT60-C629S cells expressing the hTfR, as described inMaterials and Methods. The values for CT60-WT and CT60-C629S cells weresignificantly different from that of CT60 and CT60-VEC cells (p<0.05).

FIGS. 26A, B. Decrease in cholesterol accumulation in LSOs by geneticrestoration of acid SMase activity in human fibroblasts. A) Human WT(GM05659) and NPC (GM03123) Fbs were left untransfected or weretransiently transfected with empty GFP-expressing vector (VEC) or withGFP-vector containing either WT or C629S SMPD1 cDNA constructs. Two dayslater, the cells were washed with PBS, fixed and stained with filipin.Standard UV and FITC filters were used for filipin imaging (all cells)or GFP imaging (transfected cells), respectively. The displayed filipinimages and GFP images are on the same gray scale range, respectively.Scale bar, 30 μm. B) The bar graph shows LSO ratio values normalized tothe NPC-VEC values (average of 20-30 images from three independentexperiments±SEM). The values for NPC and NPC-VEC fibroblasts weresignificantly different from those for NPC-WT and NPC-C629S fibroblasts(p<0.0001).

FIGS. 27A-C. Graph showing the effect of SMase restoration on human NPC1cells (exogenous acid SMase decreases cholesterol accumulation in LSOsin human NPC fibroblasts). Parallel sets of human WT (GM05659) and NPC(GM03123) Fbs were incubated in medium alone or, in the case of the NPCfibroblasts, medium containing 3 μg/mL recombinant human acid SMase(rhASM). Two days later, the cells were washed thoroughly with PBS andeither lysed and assayed for acid SMase activity (A), or fixed andstained with filipin for imaging and quantification (B). The images aredisplayed with the same gray scale range. Scale bar, 15 μm. Thequantified data in the bar graph represent LSO ratios normalized to theWT Fb values (average of 60-66 images from three independentexperiments±SEM). C) Another human NPC Fbs (GM18453) was incubated with3 μg/mL rhASM for 24 h, unlike 48 h in NPC1 (GM03123). Quantified datashown in bar chart are the normalized LSO ratios (normalized tountreated NPC Fbs) in the presence and absence of recombinant enzyme.Data represents an average from two independent experiments and 30-36images±SEM, p<0.0001. The values for NPC fibroblasts in panels A, B andC were significantly different from both the WT Fbs and the NPCfibroblasts treated with rhASM (p<0.0001).

FIGS. 28A-K: Addition of Alexa555-conjugated rhASM to demonstratesub-cellular localization of exogenous acid SMase. WT (GM5659) and NPC(GM03123) Fbs were incubated with or without 3 μg/mL Alexa555-conjugatedrhASM for 24 hours. To remove surface-bound label, the cells werefurther incubated for 15 min with a growth medium without the enzyme.Finally, the cells were washed with PBS, fixed with 1.5% PFA, andstained with filipin for imaging and quantification. The uptake ofrhASM-Alexa555 was completely blocked when enzyme was added in thepresence of excess mannose6-phosphate (10 mM) (data not shown). Filipinimages in panels A, D and G, and the Alexa555 images in panels B, E andH, for WT, NPC1, and NPC1+rhASM-Alexa555, respectively, are displayed onthe same gray scale range. Color overlays for filipin (green) andrhASM-Alexa555 (red) are shown in panels (C, F and I). The images in theinset are the zoomed color overlays of the region marked in (C, F andI). Scale bar=10 μm. Panel T shows the quantification of LSO filipinintensity after incubation with 0 or 3 μg/mL Alexa555conjugated rhASMfor 24 h in WT and NPC (GM03123) Fbs (values are±SEM, p<0.001).Conjugation of Alexa555 to the enzyme did not affect its activity. PanelK shows the quantification of rhASM uptake after incubation with 0 and 3μg/mL Alexa555conjugated rhASM for 24 h in WT and NPC (GM03123) Fbs(values are±SEM, p<0.05).

FIGS. 29A,B. Decreased LSO cholesterol accumulation and increased acidSMase activity are achieved in NPC1 human fibroblasts by increasingamounts of rhASM. WT (GM5659) and NPC (GM03123) Fbs were incubated with0, 0.2 and 1.8 μg/mL rhASM for 24 hours. To remove surface-bound label,the cells were further incubated for 15 minutes with growth mediumwithout the enzyme. The cells were washed thoroughly with PBS and eitherlysed and assayed for acid SMase activity (A) or fixed and stained withfilipin for imaging and quantification (B). Each data point in plot (A)is representative of three wells in an experiment, p<0.05. Plot (B)represents LSO ratios normalized to the untreated NPC Fb GM01323 values(average of 16-20 images from two independent experiments±SEM), p<0.005.

FIG. 30. Working model of how acid SMase ameliorates the traffickingdefects in NPC1 deficient cells. The primary defect in cholesteroltrafficking caused by mutant NPC1 leads to a secondary decrease in acidSMase activity. The decrease in acid SMase activity causes an increasein intracellular SM, presumably in late endosomes and possibly othersites, which amplifies the original cholesterol trafficking defect.Moreover, cholesterol accumulation in the LSO, and perhaps other effectsof NPC1 deficiency, are associated with trafficking defects in otherlipids, such as BMP, and perturbation of vesicular trafficking ofmembrane proteins, including UR recycling. Thus, these defects wouldalso be amplified by the secondary decrease in acid SMase activity.Restoring the defect in acid SMase activity breaks the amplificationcycle and thus helps correct the aforementioned lipid and proteintrafficking defects.

DETAILED DESCRIPTION OF THE INVENTION

In one aspect, the invention is directed to a composition having anactive portion therein that includes a polysaccharide having attachedthereto at least one cyclic oligosaccharide. In particular embodiments,at least one cell-targeting agent is attached to the foregoing activeportion.

In one set of embodiments, the cyclic oligosaccharide is embedded withinthe structure of the polysaccharide chain. In this embodiment, thecyclic oligosaccharide is considered to be non-pendant. A generalizedstructure depicting the foregoing embodiment is provided by thefollowing representation:

In the above representation, P₁ and P₂ each represent at least onepolysaccharide unit, wherein P₁ and P₂ are structurally the same ordifferent. C represents a cyclic oligosaccharide, or alternatively, morethan one (e.g., two, three, or more) cyclic oligosaccharides bound toeach other either directly or via one or more linkers. In particularembodiments, at least one of X₁, X₂, X₃, X₄, and X₅ represents acell-targeting agent, such as M6P. One or more of X₁, X₂, X₃, X₄, and X₅can alternatively, or in addition, (i.e., optionally) represent afluorophore, or other functional moiety, such as additional cyclicoligosaccharide groups. Each of X₁, X₂, X₃, X₄, and X₅ can alsorepresent a multiplicity (e.g., two, three, or more) of any of theforegoing groups. The dashed lines to each of X₁, X₂, X₃, X₄, and X₅indicate that these groups may or may not be present, and, if present,may be attached to any portion of the group to which they are bound.Each of the solid lines connecting P₁ and P₂ with C represents at leastone direct bond or linker. The representation in formula (1) is meant tobe non-limiting by depicting a minimum set of features that can beexpanded upon in numerous ways. For example, additional embedded cyclicoligosaccharides may be included, or additional C or P groups may bebound to any of X₁, X₂, X₃, X₄, and X₅.

In another set of embodiments, the cyclic oligosaccharide is pendant tothe polysaccharide chain. A generalized structure depicting theforegoing embodiment is provided by the following representation:

In the above representation, P represents a polysaccharide and Crepresents at least one cyclic oligosaccharide bound to P directly orvia one or more linkers. Alternatively, or in addition, C representsmore than one cyclic oligosaccharide bound to each other either directlyor via one or more linkers. In particular embodiments, at least one ofX₁, X₂, and X₃ represents a cell-targeting agent. One or more of X₁, X₂,and X₃ can alternatively, or in addition (i.e., optionally) represent afluorophore, or other functional moiety, such as additional cyclicoligosaccharide groups. In this embodiment, X₁ is not considered asanother P group, although, X₂ and/or X₃ could also represent one or moreP groups. Each of X₁, X₂, and X₃ can also represent a multiplicity(e.g., two, three, or more) of any of the foregoing groups. The dashedlines to each of X₁, X₂, and X₃ indicate that these groups may or maynot be present, and, if present, may be attached to any portion of thegroup to which they are bound. The solid line connecting P with Crepresents at least one direct bond or linker. The representation informula (2) is meant to be non-limiting by depicting a minimum set offeatures that can be expanded upon in numerous ways. For example, anadditional P group may be bound to X₂ and/or X₃, or an additional Cgroup may be bound to X₁, X₂, and/or X₃.

The cyclic oligosaccharide, as defined herein, is a chemical moietycontaining at least three monosaccharide units connected directly or viaone or more linkers to each other such that the monosaccharide units arearranged in a cyclic pattern. The number of monosaccharide units linkedin a cyclic pattern can be, for example, four, five, six, seven, eight,nine, ten, and higher numbers (e.g., up to 12, 15, 18, or 20 units). Themonosaccharide can be, for example, an aldose or a ketose, and, inaddition, either a triose, tetrose, pentose, hexose, or heptose.Typically, the monosaccharide considered herein contains at least four,five, six, or seven carbon atoms. Some specific examples ofmonosaccharides include glucose, fructose, galactose, mannose, ribose,maltose, arabinose, xylose, erythrose, xylulose, and ribulose. In oneembodiment, the cyclic oligosaccharide contains only one type ofmonosaccharide connected in a cyclic pattern. In another embodiment, thecyclic oligosaccharide contains more than one type of monosaccharide(e.g., two, three, or more) connected in a cyclic pattern. Themonosaccharide can be in a D- or L-configuration, although theD-configuration is more typical. The monosaccharide units can beconnected to each other by either an alpha (α) or beta (β) linkage, or acombination thereof, although an exclusive alpha linkage is moretypical.

In some embodiments, one or more of the monosaccharide units of thecyclic oligosaccharide can be derivatized. In one set of embodiments, atleast one of the monosaccharide units is derivatized by containing amodified hydroxyl group in which the hydrogen atom of the hydroxyl groupis replaced with a hydrocarbon group or inorganic group. Some suitabletypes of hydrocarbon groups include those containing at least one, two,three, four, five, or six carbon atoms, and which can bestraight-chained or branched, saturated or unsaturated, and cyclic oracyclic. Some particular hydrocarbon groups considered herein includemethyl, ethyl, vinyl, n-propyl, allyl, isopropyl, cyclopropyl, n-butyl,sec-butyl, isobutyl, t-butyl, cyclobutyl, 3-butenyl, n-pentyl,isopentyl, neopentyl, cyclopentyl, cyclopentenyl, n-hexyl, isohexyl,cyclohexyl, phenyl, benzyl, naphthyl, anthracenyl, phenanthrenyl, tolyl,and xylyl groups. In one embodiment, the substituting hydrocarbon groupcontains only carbon and hydrogen atoms. In another embodiment, thesubstituting hydrocarbon group includes at least one heteroatom (e.g.,at least one O, N, S, or halide atom, or combination thereof). Someexamples of heteroatom-substituted hydrocarbon groups include acylgroups (e.g., acetyl and propionyl groups), sulfonyl groups (e.g.,methylsulfonyl and tosyl groups), alkyleneoxy groups (e.g., ethylenoxygroups), alkylenehydroxy groups (e.g., —CH₂CH₂CH₂OH or —CH₂CH₂(OH)CH₃),alkyleneamino groups (e.g., —CH₂NH₂, —CH₂CH₂NH₂ or —CH₂CH₂CH₂NH₂groups), alkylenethiol groups (e.g., —CH₂CH₂CH₂SH), amido groups (e.g.,amide, N-methylamide, and N,N-dimethylamide groups), which link to thehydroxyl oxygen with the amido carbonyl to form a carbamate linkage,amino acids (e.g., a glycine, leucine, serine, or lysine group),dipeptides, oligopeptides, nucleobases (e.g., adenine, guanine,cytosine, thymine, and uracil groups), nucleosides, nucleotides,saccharides (e.g., monosaccharides, disaccharides, andoligosaccharides), lectins, cofactors, and combinations thereof, such asan alkyleneoxy-linked hydroxy, amino, amido, thiol, amino acid, peptide,or saccharide group. A substituting inorganic group can be, for example,a phosphate, diphosphate, triphosphate, phosphate ester, sulfate,sulfonate, metal ion (e.g., lithium, sodium, potassium, magnesium, orcalcium ion), or a phosphate-monosaccharide group. Furthermore, thegroup can be neutral or charged. The charged group can be cationic(e.g., an ammonium group, such as a quaternary ammonium group) oranionic (e.g., a carboxylate group).

In other embodiments, at least one of the monosaccharide units can bederivatized by having one or more hydroxyl groups therein, themselves,replaced by any of the groups described above, or by other groups, suchas an N-bound amino, N-bound amido (e.g., N-bound amide or acetylamidegroup), or a thiol group. In yet other embodiments, at least one of themonosaccharide units can be derivatized by having one or more hydroxylgroups replaced with a hydrogen atom, thereby resulting in adeoxysaccharide unit.

By methods well-known in the art, several of the groups described above,particularly those containing one or more heteroatoms (e.g., amino,amido, ester, thiol, and aldehydic groups) can be used as reactivegroups for attaching the cyclic oligosaccharide to another chemicalentity, i.e., to the polysaccharide, another cyclic oligosaccharide, acell-targeting agent, a fluorophore, or other group, either directly orvia a linker to any other these groups. Any of the other chemicalentities considered as a part of the composition herein (e.g., thepolysaccharide, cell-targeting agent, fluorophore, or other group) cancontain, or be derivatized to contain, any such reactive groups for thepurpose of attaching these groups to each other or to the cyclicoligosaccharide.

Particularly considered herein as cyclic oligosaccharides are thecyclodextrins. As is well-known in the art, cyclodextrins are typicallycomposed of five or more glucose (i.e., glucopyranoside) units connectedin a ring structure, linked as in amylose by alpha 14 (i.e., α(1→4))bonds. The cyclodextrins considered herein can conveniently berepresented by the following generic formula:

In generic formula (3) above, the R groups can be independently selectedfrom any of the groups described above, including hydrogen atom,hydrocarbon groups, heteroatom-substituted hydrocarbon groups, inorganicgroups, and biochemically-relevant groups. In some embodiments, all ofthe R groups in the formula are the same, while in other embodiments, atleast one of the R groups in the formula is chemically different fromother R groups. In further or alternative embodiments, one or more ofthe OR groups can be replaced by any of the groups described above.Furthermore, since the cyclodextrin (or, more generally, cyclicoligosaccharide) is attached to a polysaccharide, at least one (e.g.,one, two, or three) of the R groups represents either a direct bond or alinker that bonds or links, respectively, the cyclodextrin (or, moregenerally, cyclic oligosaccharide) to the polysaccharide. When genericformula (3) represents a cyclic oligosaccharide, the shown glucosegroups can be generically replaced by one or a combination of any of themonosaccharide groups described above. The subscript n denotes thenumber of monosaccharide units, and can be any number above 3, but moretypically a number of 4, 5, 6, 7, 8, 9, or 10, or a particular rangetherein. The arc shown in generic formula (3) denotes a cyclicarrangement of monosaccharide units.

Some particular cyclodextrin structures are shown in the followingsub-generic formulas:

In formulas (4), (5), and (6), R is as defined above for generic formula(3). Furthermore, any of formulas (4), (5), and (6) can genericallyrepresent any cyclic oligosaccharide with the indicated number ofsaccharide units, by generically replacing the shown glucose groups withone or a combination of any of the monosaccharide groups describedabove.

The polysaccharide, as defined herein, is a chemical moiety containing amultiplicity (e.g., at least 10, and more typically at least 100)monosaccharide units connected to each other in a linear and/or branchedarrangement. In one embodiment, the polysaccharide is ahomopolysaceharide by having all of the monosaccharide units as the sametype (e.g., all glucose units). In another embodiment, thepolysaccharide is a heteropolysaccharide by having different types ofmonosaccharide units. The polysaccharide considered herein possesses,for example, at least 10, 20, 50, 100, 200, 500, 1000, 5000, 10000,50000, 100000, 150000, 200000, or higher number of units, oralternatively, molecular weights (or average molecular weights) of atleast 100, 200, 500, 1000, 5000, 10000, 20000, 30000, 40000, 50000,60000, 70000, 80000, 90000, 100000, 150000, 200000, or 500000 Daltons(Da), or the polymer possesses a number of units or molecular weightwithin a range bounded by any of the foregoing exemplary values. One ormore of the monosaccharide units of the polysaccharide can bederivatized in the same manner as described above for the cyclicoligosaccharide. In a particular embodiment, the polysaccharide isfunctionalized with one, two, three, or higher multiplicity of aminogroups. Such a polysaccharide is denoted herein as an “aminopolysaccharide”.

Particularly considered herein are those polysaccharides constructedsolely of glucose units (i.e., a glucan polysaccharide). The glucanpolysaccharide can be an α-glucan or β-glucan polysaccharide, althoughthe glucan polysaccharide is more commonly an α-glucan polysaccharide. Aparticular class of α-glucan polysaccharide considered herein isdextran. As known in the art, a dextran generally consists predominantlyof glucose molecules linked predominantly or exclusively byα-1,6-glycosidic linkages. Depending on the type of dextran used, thedextran can contain any of a broad range of branching. The branchinggenerally results from α-1,4 glycosidic linkages, and in some cases,α-1,2 and α-1,3 glycosidic linkages. Other types of a-glucanpolysaccharide considered herein are the starches (e.g., amylase andamylopectin), glycogen, and pullulan. Some particular classes ofβ-glucan polysaccharides considered herein include cellulose,hemicellulose, cellodextrin, chrysolaminarin, lentinan, and zymosan. Insome embodiments, at least a portion of the glucose units can bederivatized, such as found in chitin (i.e., a polymer ofN-acetylglucasamine). Other derivatized glycans include theglycosaminoglycans, such as chondroitin sulfate, dermatan sulfate,heparin, heparan sulfate, hyaluronic acid, and keratan sulfate.

Other classes of polysaccharides considered herein are the fructans andgalactans. Some examples of fructans include the inulins,fructooligosaccharides, and Levan polysaccharide (a homopolysaccharideof fructose with varying degrees of branching). An example of a galactanis the class of galactooligosaccharides.

A particular example of a heteropolysaccharide considered herein is theclass of arabinoxylans, which are copolymers of arabinose and xylose.Another heteropolysaccharide considered herein is agarose.

In one set of embodiments, the polysaccharide consists solely ofmonosaccharide units. In another set of embodiments, the polysaccharidecan also include a non-saccharide moiety. For example, thepolysaccharide can be a glycoprotein or proteoglycan by containing aproteinaceous component, or the polysaccharide can be alipopolysaccharide by containing a lipid component.

According to the invention, at least one cyclic oligosaccharide iscovalently bound to the polysaccharide. In different embodiments, atleast one, two, three, four, five, six, and up to, for example, 10, 15,20, or 30 cyclic oligosaccharides are bound to the polysaccharide. Thecyclic oligosaccharide can be directly bound or linked via a linker tothe polysaccharide by any one or more suitable atoms present on thecyclic oligosaccharide. For example, in different embodiments, thecyclic oligosaccharide can be bound to the polysaccharide by one or moreof the carbon atoms of the cyclic oligosaccharide (e.g., by replacementof one of the hydroxyl groups of the cyclic oligosaccharide by a bindingatom of the polysaccharide or by a binding atom of a linker group linkedto the polysaccharide). Alternatively, the cyclic oligosaccharide can bebound to the polysaccharide by one or more of its hydroxyl groups (i.e.,oxygen atoms), or the cyclic oligosaccharide can be bound to thepolysaccharide by one or more heteroatom-containing groups present onthe cyclic oligosaccharide. In the same manner, the polysaccharide canbe directly bound or linked via a linker to the cyclic oligosaccharideby any one or more suitable atoms present on the polysaccharide.

Methods for functionalizing a polysaccharide or a cyclodextrin with alinking group are well-known in the art. See, for example, (a) Mocanu G,Vizitiu D, Carpov A. Cyclodextrin polymers. J. Bioact. Compat. Pol.,2001; 16:315-342; (b) Liu Y, Chen Y. Cooperative binding and multiplerecognition by bridged bis(β-cyclodextrin)s with functional linkers.Acc. Chem. Res., 2006; 39:681-691. (c) Ozmen E Y, Sezgin M, Yilmaz M.Synthesis and characterization of cyclodextrin-based polymers as asupport for immobilization of Candida rugosa lipase. J. Mol. Catal.B-Enzym., 2009; 57:109-114. (d) Trotta F, Cavalli R. Characterizationand applications of new hyper-cross-linked cyclodextrins. Compos.Interface, 2009; 16:39-48. For example, a linker group containing aportion reactive to a hydroxyl group (e.g., a carboxyl group, preferablyactivated by a carbodiimide) can be reacted with either the cyclodextrinor polysaccharide to form a covalent bond thereto. Alternatively, one ormore hydroxyl groups of the cyclodextrin and/or polysaccharide can beactivated by known methods (e.g., tosylation) to react with a reactivegroup (e.g., amino group) on the linker.

Since the linker links at least two chemical entities to each other, thelinker generally contains two reactive portions made to react and bondwith each chemical entity. In one embodiment, a double-reactive linkeris first attached to the cyclodextrin to produce a linker-cyclodextrincompound that is isolated, and then the remaining reactive portion ofthe linker in the linker-cyclodextrin compound is subsequently reactedwith the polysaccharide. In another embodiment, a double-reactive linkeris first attached to the polysaccharide to produce alinker-polysaccharide compound that is isolated, and then the remainingreactive portion of the linker in the linker-polysaccharide compound issubsequently reacted with the cyclodextrin. In the two foregoingembodiments, the second reactive portion of the linker is generallyprotected during reaction of the first reactive group, or alternatively,protection is not necessary in an embodiment where the first and secondreactive portions of the linker react with the cyclodextrin differentlythan with the polysaccharide. Particularly by the latter embodiment, adouble-reactive linker may be reacted with both the cyclodextrin andpolysaccharide simultaneously to link these groups together. In otherembodiments, the linker can have additional reactive groups in order tolink to additional cyclodextrin or polysaccharide groups, or to link toa cell-targeting and/or other functional group, such as a fluorophore.

Numerous double-reactive linkers are known in the art. Such linkers canbe used for linking any of a variety of groups together when the groupspossess, or have been functionalized to possess, groups that can reactand link with the reactive linker. Some groups capable of reacting withdouble-reactive linkers include amino, thiol, hydroxyl, carboxyl, ester,and alkyl halide groups. For example, amino-amino coupling reagents canbe employed to link a cyclic oligosaccharide with a polysaccharide (or,for example, any of these groups with a fluorophore or with each other)when each of the groups to be linked possess at least one amino group.Some examples of amino-amino coupling reagents include diisocyanates,alkyl dihalides, dialdehydes, disuccinimidyl suberate (DSS),disuccinimidyl tartrate (DST), and disulfosuccinimidyl tartrate(sulfa-DST), all of which are commercially available. In otherembodiments, amino-thiol coupling agents can be employed to link a thiolgroup of one molecule with an amino group of another molecule. Someexamples of amino-thiol coupling reagents include succinimidyl4-(N-maleimidomethyl)-cyclohexane-1-carboxylate (SMCC), andsulfosuccinimidyl 4-(N-maleimidomethyl)-cyclohexane-1 -carboxylate(sulfa-SMCC), In yet other embodiments, thiol-thiol coupling agents canbe employed to link groups bearing at least one thiol group.

The linker considered herein is any group that can link at least onecyclic oligosaccharide with at least one polysaccharide. In oneembodiment, a linker links one cyclic oligosaccharide to onepolysaccharide. In another embodiment, a linker links one cyclicoligosaccharide to two or more polysaccharides. In another embodiment, alinker links two or more cyclic oligosaccharides to one polysaccharide.In yet another embodiment, a linker links two or more cyclicoligosaccharides to two or more polysaccharides. In further embodimentsto any of the above embodiments, the linker may also link to acell-targeting agent and/or a fluorophore and/or other chemical moiety.In some embodiments, a first linker links the polysaccharide with thecyclic oligosaccharide, and a separate second linker links acell-targeting agent with the polysaccharide or the cyclicoligosaccharide. In other embodiments, a linker that links thepolysaccharide with the cyclic oligosaccharide also links with acell-targeting agent. In further embodiments to any of the foregoingembodiments, a separate third linker may be employed to link afluorophore or other functional group with the polysaccharide or thecyclic oligosaccharide, or alternatively, the first or second linkerdescribed above may also link to a fluorophore or other functionalentity. The other functional entity can be, for example, a drug orprodrug that would be hydrolyzed and/or otherwise released upon reachingthe targeted cell.

In some embodiments, the linker is as small as a single atom (e.g., an—O—, —CH₂—, or —NH— linkage), or two or three atoms in length (e.g., anamido, ureido, carbamato, ester, carbonate, sulfone, ethylene, ortrimethylene linkage). In other embodiments, the linker provides morefreedom of movement by being at least four, five, six, seven, or eightatom lengths, and up to, for example, 10, 12, 14, 16, 18, 20, 22, 24,26, 28, or 30 atom lengths.

In one embodiment, the linker is a hydrocarbon linker, e.g., as derivedfrom any of the hydrocarbon groups described above by removal of two ormore hydrogen atoms therefrom (thus resulting in two or more linkingbonds therein). Some examples of hydrocarbon linkers include methylene(—CH₂—), ethylene (—CH₂CH₂‘3), trimethylene (—CH₂CH₂CH₂—),tetramethylene, pentamethylene, hexamethylene, o-, and p-phenylene, andvinylene.

The hydrocarbon linker may or may not also include heteroatoms.Furthermore, the heteroatoms may or may not be linking atoms. In aparticular embodiment, the hydrocarbon linker contains one, two, three,or more amino groups. Some examples of amino-containing linkers include1,2-ethylenediamine, 1,3-trimethylenediamine, 1,4-tetramethylenediamine,1,5-pentamethylenediamine, 1,6-hexamethylenediamine, diethylenetriamine,triethylenetetramine, and diaminobenzene linkers. In another particularembodiment, the hydrocarbon linker contains one, two, three, or moreoxygen-linking (i.e., —O—) atoms or hydroxy groups. Some examples ofsuch linkers include ethylene glycol, diethylene glycol, triethyleneglycol, 2-hydroxypropane, 2,3-dihydroxybutane, dihydroxybenzene, and thepolyethylene glycol (i.e., PEG) linkers. In other particularembodiments, the linkers include one, two, three, or more carbonylgroups. Some examples of such linkers include methyl dicarbonyl,ethylene-1,2-dicarbonyl, propylene-1,3-dicarbonyl, and the like.

The linker can also be or include a biological group, such as, forexample, a nucleobase, nucleoside, nucleotide, dinucleotide,trinucleotide, tetranucleotide, a higher oligonucleotide, amino acid,dipeptide, tripeptide, tetrapeptide, pentapeptide, hexapeptide, a higheroligopeptide, saccharide, disaccharide, trisaccharide, tetrasaccharide,a higher oligosaccharide, lipid, or fatty acid.

In particular embodiments, the linker is a rigid linker. A rigid linkermay be beneficial in some embodiments by reducing the degree of freedomof a linked molecule, or forcing at least two linked groups to remain atfixed distances from each other or from another molecule. Some examplesof rigid linkers are those containing aromatic or heteroaromatic rings,such as linkers that include benzene, naphthalene, styrene,divinylbenzene, biphenyl, triphenyl, or other aromatic rings orpolycyclic ring systems.

In a particular set of embodiments, the linker has a structurerepresented by the following generic formula:

In formula (7) above, subscripts r, s, and t can independently be 0 oran integer of at least 1, provided that at least one of r, s, and t isnot 0. Generally, each end of the linker generally also includes aheteroatom-containing group through which a covalent bond is formedbetween the linker and groups that are linked. As discussed earlier,such heteroatom-containing groups include, for example, oxo (—O—), amino(e.g., —NH—, or —N(CH₃)—), amido (e.g., —C(O)NH— or —C(O)N(CH₃)—),ester, ureido, carbonato, sulfanoto, and phosphonato groups. Indifferent embodiments, r, s, and t are independently selected from 0, oran integer of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, or 16,or selected to be within a range therein, provided that at least one ofr, s, and t is not 0. In one set of embodiments, r and t are 0, and srepresents any of the foregoing non-zero numbers or possible rangestherein. In another set of embodiments, r is 0, while s and tindependently represent any of the foregoing non-zero numbers orpossible ranges therein. In another set of embodiments, s is zero whileat least one of r and t represents any of the foregoing non-zero numbersor possible ranges (thereby resulting in an alkylene structure forformula 7). In another set of embodiments, r, s, and t independentlyrepresent any of the foregoing non-zero numbers or possible rangestherein. In particular embodiments, r and t are independently 1, 2, 3,or 4, or a subset therein, while s is selected from any of the foregoingnon-zero numbers or possible ranges therein.

The cell-targeting agent (i.e., “targeting agent”) is any chemicalentity that has the ability to bind to (i.e., “target”) a cell. The cellparticularly considered herein is a mammalian cell. The type of cell canbe any cell in which cholesterol, a cholesterol derivative, or otherlipid can accumulate. Some examples of types of cells that can betargeted include cells of the blood, bone marrow, spleen, liver, skin,lungs, nerves (particularly of the peripheral nervous system), andbrain. The cell-targeting agent may target any part of the cell, e.g.,cell membrane, organelle, or cytoplasmic molecule. In one embodiment,the cell-targeting agent targets a cell in a selective manner. Byselectively targeting a cell, the cell-targeting agent can, for example,selectively target certain types of cells over other types of cells, ortarget certain parts of a cell over other parts of a cell, or both. Inother embodiments, the targeting agent targets cells non-selectively,e.g., by targeting components found in most or all cells.

In various embodiments, the targeting agent can be, or include, forexample, a peptide, dipeptide, tripeptide (e.g., glutathione),tetrapeptide, pentapeptide, hexapeptide, higher oligopeptide, protein,monosaccharide, disaccharide, trisaccharide, tetrasaccharide, higheroligosaccharide, polysaccharide (e.g., a carbohydrate), nucleobase,nucleoside (e.g., adenosine, cytidine, uridine, guanosine, thymidine,inosine, and S-Adenosyl methionine), nucleotide (i.e., mono-, di-, ortri-phosphate forms), dinucleotide, trinucleotide, tetranucleotide,higher oligonucleotide, nucleic acid, cofactor (e.g., TPP, FAD, NAD,coenzyme A, biotin, lipoamide, metal ions (e.g., Mg²⁺), metal-containingclusters {e.g., the iron-sulfur clusters), or a non-biological (i.e.,synthetic) targeting group. Some particular types of proteins includeenzymes, hormones, antibodies (e.g., monoclonal antibodies), lectins,and steroids.

Antibodies for use as targeting molecules are generally specific for oneor more cell surface antigens. In a particular embodiment, the antigenis a receptor. The antibody can be a whole antibody, or alternatively, afragment of an antibody that retains the recognition portion (i.e.,hypervariable region) of the antibody. Some examples of antibodyfragments include Fab, Fe, and F(ab′)₂. In particular embodiments,particularly for the purpose of facilitating crosslinking of theantibody to the composition described herein, the antibody or antibodyfragment can be chemically reduced to derivatize the antibody orantibody fragment with sulthydryl groups.

In particular embodiments, the targeting agent is a ligand of aninternalized receptor of the target cell. For example, the targetingagent can be a targeting signal for acid hydrolase precursor proteinsthat transport various materials to lysosomes. One such targeting agentof particular interest is mannose-6-phosphate (MGP), which is recognizedby mannose 6-phosphate receptor (MPR) proteins in the trans-Golgi.Endosomes are known to be involved in transporting M6P-labeledsubstances to lysosomes.

In another embodiment, the targeting molecule is a peptide containing anRGD sequence, or variants thereof, that bind RGD receptors on thesurface of many types of cells. Other ligands include, for example,transferrin, insulin, amylin, and the like. Receptor internalization ispreferred to facilitate intracellular delivery of the inventivecomposition described herein.

In one set of embodiments, one cell-targeting molecule, or several(e.g., two, three, or more) of the same type of cell-targeting moleculeare attached to the inventive composition (particularly on the activeportion therein). In other embodiments, two or more different types oftargeting molecules are attached to the inventive composition. At leastone advantage in using several cell-targeting molecules is that uptakeof the inventive composition into cells is generally increased relativeto use of a single cell-targeting molecule.

In some embodiments, a fluorophore may be attached to the compositiondescribed above. Incorporation of one or more fluorophores can haveseveral purposes, but at least in some embodiments, one or morefluorophores are included in order to quantify cellular uptake andretention of the above-described composition (e.g., by a fluorescencespectroscopic method).

As used herein, a “fluorophore” refers to any species with the abilityto fluoresce (i.e., that possesses a fluorescent property). For example,in one embodiment, the fluorophore is an organic fluorophore. Theorganic fluorophore can be, for example, a charged (i.e., ionic)molecule (e.g., sulfonate or ammonium groups), uncharged (i.e., neutral)molecule, saturated molecule, unsaturated molecule, cyclic molecule,bicyclic molecule, tricyclic molecule, polycyclic molecule, acyclicmolecule, aromatic molecule, and/or heterocyclic molecule (i.e., bybeing ring-substituted by one or more heteroatoms selected from, forexample, nitrogen, oxygen and sulfur). In the particular case ofunsaturated fluorophores, the fluorophore contains one, two, three, ormore carbon-carbon and/or carbon-nitrogen double and/or triple bonds. Ina particular embodiment, the fluorophore contains at least two (e.g.,two, three, four, five, or more) conjugated double bonds aside from anyaromatic group that may be in the fluorophore. In other embodiments, thefluorophore is a fused polycyclic aromatic hydrocarbon (PAH) containingat least two, three, four, five, or six rings (e.g., naphthalene,pyrene, anthracene, chrysene, triphenylene, tetracene, azulene, andphenanthrene) wherein the PAH can be optionally ring-substituted orderivatized by one, two, three or more heteroatoms orheteroatom-containing groups.

In other embodiments, the organic fluorophore is a xanthene derivative(e.g., fluorescein, rhodamine, Oregon green, eosin, and Texas Red),cyanine or its derivatives or subclasses (e.g., streptocyanines,hemicyanines, closed chain cyanines, phycocyanins, allophycocyanins,indocarbocyanines, oxacarbocyanines, thiacarbocyanines, merocyanins, andphthalocyanines), naphthalene derivatives (e.g., dansyl and prodanderivatives), coumarin and its derivatives, oxadiazole and itsderivatives (e.g., pyridyloxazoles, nitrobenzoxadiazoles, andbenzoxadiazoles), pyrene and its derivatives, oxazine and itsderivatives (e.g., Nile Red, Nile Blue, and cresyl violet), acridinederivatives (e.g., proflavin, acridine orange, and acridine yellow),arylmethine derivatives (e.g., auramine, crystal violet, and malachitegreen), and tetrapyrrole derivatives (e.g., porphyrins and bilirubins).Some particular families of dyes considered herein are the Cy® family ofdyes, the Alexa® family of dyes, the ATTO® family of dyes, and the Dy®family of dyes. The ATTO dyes, in particular, can have severalstructural motifs, including, coumarin-based, rhodamine-based,carbopyronin-based, and oxazine-based structural motifs.

The fluorophore can be attached to the active portion (e.g., to thecyclic oligosaccharide, polysaccharide, a linker, or other group) by anyof the linking methodologies known in the art. For example, a commercialmono-reactive fluorophore (e.g., NHS-Cy5) or bis-reactive fluorophore(e.g., bis-NHS-Cy5 or bis-maleimide-Cy5) can be used to link thefluorophore to one or more molecules containing appropriate reactivegroups (e.g., amino, thiol, hydroxy, aldehydic, or ketonic groups).Alternatively, the active portion of the inventive composition can bederivatized with one, two, or more such reactive groups, and thesereactive portions reacted with a fluorophore containing appropriatereactive groups (e.g., an amino-containing fluorophore).

In another aspect, the invention is directed to a cholesterol-loweringcomposition that includes a histone deacetylase (HDAC) inhibitingcompound. In a first embodiment, the HDAC-inhibiting compositionincludes trichostatin A (TSA) as a cholesterol-lowering activeingredient. In a second embodiment, the HDAC-inhibiting compositionincludes suberoylanilide hydroxamic acid (SAHA) as acholesterol-lowering active ingredient. In a third embodiment, theHDAC-inhibiting composition includes pyroxamide as acholesterol-lowering active ingredient. In a fourth embodiment, theHDAC-inhibiting composition includes C1-994 (N-Acetyldinaline, asubstituted benzamide derivative, i.e. 4-acetylamino-N-(2-aminophenyl)benzamide) as a cholesterol-lowering active ingredient. In a fifthembodiment, the HDAC-inhibiting composition includes Bufexamac (i.e.,2-(4-butoxyphenyl)-N-hydroxyacetamide) as a cholesterol-lowering activeingredient. In a sixth embodiment, the HDAC-inhibiting compositionincludes LBH-589 (i.e. Panobinostat) as a cholesterol-lowering activeingredient. In other embodiments, the HDAC-inhibiting compositionincludes two or more of any of the foregoing cholesterol-loweringcompounds. In yet other embodiments, one or more of the HDAC-inhibitingcompounds are combined with the cyclic oligosaccharide-polysaccharidecomposition described above as a pharmaceutical composition for loweringcholesterol in a mammal.

In another aspect, the invention is directed to a cholesterol-loweringcomposition that includes a sphingomyelinase (SMase). As known in theart, cholesterol-enriched NPC cells have a secondary, post-translationaldefect in the activity of another lysosomal enzyme, acid SMase (J. W.Reagan, Jr., et al., Posttranslational regulation of acidsphingomyelinase in Niemann Pick type C1 fibroblasts and freecholesterol-enriched Chinese hamster ovary cells, J. Biol. Chem., 275(2000) pp. 38104-38110). The resulting lysosomal accumulation ofsphingomyelin (SM) appears to stabilize cholesterol in the LSOs, therebyexacerbating the cholesterol accumulation. To counteract this effect,this aspect of the invention is directed to at least partially restoringSMase activity in an effort to reduce cholesterol accumulation. In otherembodiments, the foregoing SMase composition is combined with the cyclicoligosaccharide-polysaccharide composition described above as apharmaceutical composition for lowering cholesterol in a mammal.

In another aspect, the invention is directed to a pharmaceuticalcomposition that contains any of the above cholesterol-loweringcompositions in a pharmaceutically acceptable vehicle (i.e., excipient).The pharmaceutical composition can also be formulated together with oneor more medications that improves the overall efficacy of thepharmaceutical composition and/or reduces or avoid side effects.

The active ingredient(s) and excipient(s) may be formulated intocompositions and dosage forms according to methods known in the art. Asdescribed in detail below, the pharmaceutical compositions of thepresent invention may be specially formulated for administration insolid or liquid form, including those adapted for the following: (1)oral administration, for example, tablets, capsules, powders, granules,pastes for application to the tongue, aqueous or non-aqueous solutionsor suspensions, drenches, or syrups; (2) parenteral administration, forexample, by subcutaneous, intramuscular or intravenous injection as, forexample, a sterile solution or suspension; (3) topical application, forexample, as a cream, ointment or spray applied to the skin, lungs, ormucous membranes; or (4) intravaginally or intrarectally, for example,as a pessary, cream or foam; (5) sublingually or buccally; (6) ocularly;(7) transdermally; or (8) nasally.

The phrase “pharmaceutically acceptable” refers herein to thosecompounds, materials, compositions, and/or dosage forms which are,within the scope of sound medical judgment, suitable for administrationto a subject. The phrase “pharmaceutically acceptable excipient” as usedherein refers to a pharmaceutically acceptable material, composition orvehicle, such as a liquid or solid filler, diluent, carrier,manufacturing aid (e.g., lubricant, talc magnesium, calcium or zincstearate, or steric acid), solvent or encapsulating material, involvedin carrying or transporting the therapeutic composition foradministration to the subject. Each excipient should be “acceptable” inthe sense of being compatible with the other ingredients of theformulation and not injurious to the subject.

Some examples of materials which can serve as pharmaceuticallyacceptable excipients, particularly for liquid forms, include sugars(e.g., lactose, glucose and sucrose); starches (e.g., corn and potatostarch); cellulose and its derivatives (e.g., sodium carboxymethylcellulose, ethyl cellulose and cellulose acetate); gelatin; talc; waxes;oils (e.g., peanut oil, cottonseed oil, safflower oil, sesame oil, oliveoil, corn oil and soybean oil); glycols (e.g., ethylene glycol,propylene glycol, and polyethylene glycol); polyols (e.g., glycerin,sorbitol, and mannitol); esters (e.g., ethyl oleate and ethyl laurate);agar; buffering agents; water; isotonic saline; pH buffered solutions;and other non-toxic compatible substances employed in pharmaceuticalformulations. If desired, certain sweetening and/or flavoring and/orcoloring agents may be added. Other suitable excipients can be found instandard pharmaceutical texts, e.g. in “Remington's PharmaceuticalSciences”, The Science and Practice of Pharmacy, 19th Ed. MackPublishing Company, Easton, Pa., (1995).

Diluents increase the bulk of a solid pharmaceutical composition, andmay make a pharmaceutical dosage form that is easier for the patient andcaregiver to handle. Diluents for solid compositions include, forexample, microcrystalline cellulose (e.g. Avicel®), microfine cellulose,lactose, starch, pregelatinized starch, calcium carbonate, calciumsulfate, sugar, dextrates, dextrin, dextrose, dibasic calcium phosphatedihydrate, tribasic calcium phosphate, kaolin, magnesium carbonate,magnesium oxide, maltodextrin, mannitol, polymethacrylates (e.g.Eudragit®), potassium chloride, powdered cellulose, sodium chloride,sorbitol and talc.

Solid pharmaceutical compositions that are compacted into a dosage form,such as a tablet, may include excipients whose functions include helpingto bind the active ingredient and other excipients together aftercompression. Binders for solid pharmaceutical compositions includeacacia, alginic acid, carbomer (e.g. carbopol), carboxymethylcellulosesodium, dextrin, ethyl cellulose, gelatin, guar gum, hydrogenatedvegetable oil, hydroxyethyl cellulose, hydroxypropyl cellulose (e.g.Klucel®), hydroxypropyl methyl cellulose (e.g. Methocel®), liquidglucose, magnesium aluminum silicate, maltodextrin, methylcellulose,polymethacrylates, povidone (e.g. Kollidon®, Plasdone®), pregelatinizedstarch, sodium alginate and starch.

The dissolution rate of a compacted solid pharmaceutical composition inthe subject's stomach may be increased by the addition of a disintegrantto the composition. Disintegrants include alginic acid,carboxymethylcellulose calcium, carboxymethylcellulose sodium (e.g. AcDi Sol®, Primellose®), colloidal silicon dioxide, croscarmellose sodium,crospovidone (e.g. Kollidon®, Polyplasdone®), guar gum, magnesiumaluminum silicate, methyl cellulose, microcrystalline cellulose,polacrilin potassium, powdered cellulose, pregelatinized starch, sodiumalginate, sodium starch glycolate (e.g. Explotab®) and starch.

Glidants can be added to improve the flowability of a non-compactedsolid composition and to improve the accuracy of dosing. Excipients thatmay function as glidants include colloidal silicon dioxide, magnesiumtrisilicate, powdered cellulose, starch, talc and tribasic calciumphosphate.

When a dosage form such as a tablet is made by the compaction of apowdered composition, the composition is subjected to pressure from apunch and dye. Some excipients and active ingredients have a tendency toadhere to the surfaces of the punch and dye, which can cause the productto have pitting and other surface irregularities. A lubricant can beadded to the composition to reduce adhesion and ease the release of theproduct from the dye. Lubricants include, for example, magnesiumstearate, calcium stearate, glyceryl monostearate, glycerylpalmitostearate, hydrogenated castor oil, hydrogenated vegetable oil,mineral oil, polyethylene glycol, sodium benzoate, sodium laurylsulfate, sodium stearyl fumarate, stearic acid, talc and zinc stearate.

In liquid pharmaceutical compositions of the present invention, themodulator of bacterial adenyl cyclase and any other solid excipients aredissolved or suspended in a liquid carrier such as water,water-for-injection, vegetable oil, alcohol, polyethylene glycol,propylene glycol or glycerin.

Liquid pharmaceutical compositions may contain emulsifying agents todisperse uniformly throughout the composition an active ingredient orother excipient that is not soluble in the liquid carrier. Emulsifyingagents that may be useful in liquid compositions of the presentinvention include, for example, gelatin, egg yolk, casein, cholesterol,acacia, tragacanth, chondrus, pectin, methylcellulose, carbomer,cetostearyl alcohol and cetyl alcohol.

Liquid pharmaceutical compositions of the present invention may alsocontain a viscosity-enhancing agent to improve the mouthfeel of theproduct and/or coat the lining of the gastrointestinal tract. Suchagents include acacia, alginic acid bentonite, carbomer,carboxymethylcellulose calcium or sodium, cetostearyl alcohol, methylcellulose, ethylcellulose, gelatin guar gum, hydroxyethyl cellulose,hydroxypropyl cellulose, hydroxypropyl methyl cellulose, maltodextrin,polyvinyl alcohol, povidone, propylene carbonate, propylene glycolalginate, sodium alginate, sodium starch glycolate, starch tragacanthand xanthan gum.

Sweetening agents, such as sorbitol, saccharin, sodium saccharin,sucrose, aspartame, fructose, mannitol and invert sugar, may be added toimprove the taste. Flavoring agents and flavor enhancers may make thedosage form more palatable to the patient. Common flavoring agents andflavor enhancers for pharmaceutical products that may be included in thecomposition of the present invention include, for example, maltol,vanillin, ethyl vanillin, menthol, citric acid, fumaric acid, ethylmaltol and tartaric acid.

Preservatives and chelating agents, such as alcohol, sodium benzoate,butylated hydroxy toluene, butylated hydroxyanisole and ethylenediaminetetraacetic acid, may be added at levels safe for ingestion to improvestorage stability.

According to the present invention, a liquid composition may alsocontain a buffer such as gluconic acid, lactic acid, citric acid oracetic acid, sodium gluconate, sodium lactate, sodium citrate or sodiumacetate. Selection of excipients and the amounts used may be readilydetermined by the formulation scientist based upon experience andconsideration of standard procedures and reference works in the field.

Solid and liquid compositions may also be dyed using anypharmaceutically acceptable colorant to improve their appearance and/orfacilitate patient identification of the product and unit dosage level.

The dosage form of the present invention may be a capsule containing thecomposition, for example, a powdered or granulated solid composition ofthe invention, within either a hard or soft shell. The shell may be madefrom gelatin and optionally contain a plasticizer such as glycerin andsorbitol, and an opacifying agent or colorant.

A composition for tableting or capsule filling may be prepared by wetgranulation. In wet granulation, some or all of the active ingredientsand excipients in powder form are blended and then further mixed in thepresence of a liquid, typically water, that causes the powders to clumpinto granules. The granulate is screened and/or milled, dried and thenscreened and/or milled to the desired particle size. The granulate maythen be tableted, or other excipients may be added prior to tableting,such as a glidant and/or a lubricant.

A tableting composition may be prepared conventionally by dry blending.For example, the blended composition of the actives and excipients maybe compacted into a slug or a sheet and then comminuted into compactedgranules. The compacted granules may subsequently be compressed into atablet.

As an alternative to dry granulation, a blended composition may becompressed directly into a compacted dosage form using directcompression techniques. Direct compression produces a more uniformtablet without granules. Excipients that are particularly well suitedfor direct compression tableting include microcrystalline cellulose,spray dried lactose, dicalcium phosphate dihydrate and colloidal silica.The proper use of these and other excipients in direct compressiontableting is known to those in the art with experience and skill inparticular formulation challenges of direct compression tableting.

A capsule filling may include any of the aforementioned blends andgranulates that were described with reference to tableting, except thatthey are not subjected to a final tableting step.

In another aspect, the invention is directed to methods for lowering acholesterol level of a mammal by administering to the mammal acholesterol-reducing composition described above. The mammal primarilyconsidered herein is a human, although other mammals, such as dogs,cats, monkeys, cows, and others, can benefit as well.

In one embodiment, the composition is administered to the mammal in sucha manner that the composition does not specifically target particulartissue or cells of the body. In the foregoing embodiment, the overallcholesterol level of the mammal is reduced. In another embodiment, thecomposition is administered to the mammal in such a manner that thecomposition selectively targets particular tissue or cells of the body.The composition can be made to selectively target particular tissue orcells within a mammal by, for example, administering the composition ina localized manner at the site of target tissue or cells (e.g., byinjection into target tissue or cells). In an alternative embodiment,the composition can be made to selectively target particular tissue orcells within a mammal by administering the composition non-locally orlocally, and including in the composition a selective targeting agentthat selectively targets certain tissues or certain cells of the body(e.g., by employing an antibody targeting agent). The tissue beingtreated can be, for example, tissue of the liver, bone marrow, spleen,skin, lungs, nerves (particularly of the peripheral nervous system), andbrain. The end result of the therapy is that the mammal experiences,either overall or in specific treated tissue, a reduced amount ofcholesterol.

As described above, and depending on how the composition is formulatedas well as the type of condition to be treated, the composition can beadministered orally (e.g., by swallowing or enteral ingestion oftablets, capsules, powders, granules, pastes, solutions, suspensions,drenches, or syrups); parenterally, by, for example, subcutaneous,intramuscular or intravenous injection as; topically, by, for example,applying as a cream, ointment or spray to the skin, internal organs(e.g., lungs), or mucous membranes; or by applying as a pessary, creamor foam, or sublingually; ocularly, transdermally, or nasally.

In order to realize the therapeutic effect of a reduced cholesterollevel, the cholesterol-lowering composition is administered in atherapeutically-effective amount. As is well known in the art, thedosage of the active ingredient(s) significantly depends on such factorsas the extent of the lipid (e.g., cholesterol) accumulation, method ofadministration, size of the patient, and potential side effects. Indifferent embodiments, depending on these and other factors, a suitabledosage of the active ingredient of the cholesterol-lowering compositionmay be precisely, at least, or no more than, for example, 1 mg, 10 mg,50 mg, 100 mg, 200 mg, 300 mg, 400 mg, 500 mg, 600 mg, 700 mg, 800 mg,900 mg, 1000 mg, 1200 mg, or 1500 mg, or a dosage within a range boundedby any of the foregoing exemplary dosages. Further to the aboveembodiments, depending on the same and other factors, the composition isadministered in the indicated amount by any suitable schedule, e.g.,once, twice, or three times a day for a total treatment time of one,two, three, four, or five days, and up to, for example, one, two, three,or four weeks. Alternatively, or in addition, the composition isadministered until a desired cholesterol level is reached. The desiredcholesterol level can be any cholesterol level deemed by a professionalin the medical arts to be appropriate to achieve.

The method described herein can be used to treat any conditionexpressing itself by an elevated level of a lipid in one or more tissuesof a mammal. In one set of embodiments, the condition is a non-inheritedform of hypercholesteremia caused primarily by environmental causes suchas poor diet and/or other unhealthy lifestyle choices (e.g., lack ofexercise and/or smoking). In another set of embodiments, the conditionis inherited, as found in the lysosomal storage diseases (LSDs). The LSDbeing treated can be, for example, Niemann-Pick disease, Gaucherdisease, GM1 gangliosidoses, GM2 gangliosidoses, Fabry disease, Krabbedisease, fucosidosis, metachromatic leukodystrophy (MLD), Wolmandisease, Farber disease, or Schindler disease.

Examples have been set forth below for the purpose of illustration andto describe the best mode of the invention at the present time. However,the scope of this invention is not to be in any way limited by theexamples set forth herein.

Furthermore, the complete disclosure of A. I. Rosenbaum, et al. PNAS,vol. 107, no. 12 (Mar. 23, 2010), pp. 5477-5482, including all exemplaryinformation, discussion, and supporting information found therein, isherein incorporated by reference in its entirety.

EXAMPLE 1 Synthesis of Cyclodextrin (CD) Polymers

General Description

The synthesis of the methyl β-cyclodextrin-PEG-dextran conjugate wasdesigned to be fully modular such that any single piece can be modifiedwithout having to completely change the overall procedure. The PEGgroups are incorporated onto the dextran polymer by amide formationbetween commercially available aminodextran and an appropriatelyprotected amino-PEG-acid (e.g., BocHN-(PEG)_(n)-CO₂H). Removal of theN-protecting group then liberates an amine that can be coupled directlyto methyl β-cyclodextrin via direct S_(N)2 displacement of the 1° tosylgroup on 6^(A)-O-p-toluenesulfonyl-permethyl-β-cyclodextrin. If thisfinal coupling is not run to completion, the remaining free amines canbe used to conjugate a second molecule of interest (e.g., a fluorescenttag or a sugar).

Synthesis of Methyl-β-Cyclodextrin(MbCD or MβCD) Conjugate

Synthesis of mono(6A-O-p-toluenesulfonyl)-β-cyclodextrin(i.e.,“tosylated β-cyclodextrin”). 1.2 g of -β-Cyclodextrin wassuspended in 10 mL H₂O, and 0.4 mL of NaOH aqueous solution (1.65 g in 5mL H₂O) was added dropwise. After ˜10 minutes, p-tosyl chloride wasadded gradually over about 10 minutes. Immediate precipitation wasobserved. The reaction was continued at ambient temperature (i.e.,generally within 15-30° C.) for another two hours. The resultingsolution was filtered, and the precipitate collected. The precipitatewas dried under high vacuum in the presence of P₂O₅ overnight. Theproduct at this stage is tosylated β-cyclodextrin.

Synthesis of mono(6A-O-p-toluenesulfonyl)permethyl-β-cyclodextrin (i.e.,“permethylated tosylated β-cyclodextrin” or “tosyl-MβCD”). Tosylatedβ-cyclodextrin (0.8 g), produced as above, was dissolved in DMF and theresulting solution cooled to 0° C. After 10 minutes, 1.8 g of sodiumhydride was added. The reaction mixture was stirred at the sametemperature for one hour before it was warmed to ambient temperature andstirred for another hour. The mixture was then cooled to 0° C. Then 12.5g of iodomethane was added. The reaction mixture was stirred at 0° C.for one hour, and then gradually warmed to ambient temperature. Theresulting mixture was stirred for about 24 hours. After the reaction wascomplete, the mixture was poured into ice-water, and then extracted withCHCl₃. The organic layer was washed with sodium thiosulfate, water, andbrine, sequentially. The volatiles were removed in vacuo and the crudeproduct purified with 0.1-1% of methanol in ethyl acetate.

Synthesis of Boc-NH-(PEG)₃-CONH-aminodextran. Separately, 500 mg ofaminodextran (MW of 70,000) was suspended in 10.0 mL DMF. Theaminodextran used generally contains a multiplicity of amine groups.After 5 minutes, 0.4 mL (5 mmol) of pyridine was added and the mixturestirred at ambient temperature for 10 minutes. At that point, ca. 510 mg(1.4 mmol) of Boc-NH-(PEG)₃-COOH and ca. 540 mg of1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC) were sequentiallyadded. The resulting mixture was vigorously stirred at ambienttemperature overnight (i.e., ca. 18 hours) and then purified byextensive dialysis against water (Spectra/Por tubing, MW cutoff of 12-14kDa) to remove unreacted small molecules. Finally, the product was twicelyophilized to furnish the PEGylated aminodextran as a white powder (ca.600 mg). If storage was necessary, the product was stored in arefrigerator at −20° C.

Synthesis of NH₂-(PEG)₃-CONH-aminodextran. Boc-NH-(PEG)₃-CONH-dextran(ca. 600 mg), produced as above, was added to a flask containing 15 mLof trifluoroacetic acid (TPA) at 0° C., and the mixture stirred for 30minutes, at which time the mixture was gradually warmed to ambienttemperature and stirred for an additional 45 minutes. The resultingcrude product was diluted with water and purified by dialysis (e.g.,three times) against water. After lyophilization (3×), the product wasobtained as a white powder (ca. 600 mg).

Synthesis of MβCD-NH-(PEG)₃-CONH-aminodextran. NH₂-(PEG)₃-CONH-dextran(ca. 600 mg), produced as described above, was dissolved in 45 mL offormamide. To this solution was added 560 mg (0.4 mmol) of permethylatedtosylated β-cyclodextrin, prepared as above. The reaction mixture washeated to 100° C. and stirred for 48 hours. Completion of the reactionwas confirmed by TLC. The reaction mixture was then diluted with waterand extensively dialyzed against water. After lyophilization (twice),the product was obtained as a white powder (530 mg). The product wasstored in a −20° C. refrigerator and could be used without furtherpurification.

Synthesis of Alexafluor546-MβCD-NH-(PEG)₃-CONH-aminodextran.MβCD-NH-(PEG)₃-CONH-aminodextran (ca. 530 mg) was added to a minimalamount of phosphate-buffered saline (PBS) solution, and the mixturestirred at ambient temperature for 1 hour. At that point, AlexaFluor546NHS ester (0.4 mg, 0.37 μmol) was added. The reaction mixture wasstirred at ambient temperature for an additional 48 hours. Afterextensive dialysis against water, and lyophilization (3×), a pinkcotton-like solid product was obtained (427 mg). The final product wasstored in a −20° C. refrigerator before used in a cell assay.

The synthesis described herein easily allows for the incorporation ofother adducts, such as mannose-6-phosphate (M6P). The synthesisdescribed herein also allows for variation, modification, oroptimization of the size of the dextran, the size of the PEG linker tothe MβCD, and the degree of substitution with both M6P and MβCD, all ofwhich have an impact on the efficacy of the composition. Furthermore,the density of M6P affects the avidity of binding by altering theeffective valency of the ligand-receptor interaction. Since two MβCDsare presumably required to solubilize a cholesterol molecule, thedensity of MβCD is important (i.e., high density of MβCD promotescholesterol binding).

EXAMPLE 2 Use of Cyclodextrins to Reduce Cholesterol

The efficacy of various MβCD-polymers (and other adducts ormodifications of MβCD) in human NPC fibroblasts was tested. The two celllines tested herein are two NPC1 lines and one or two NPC2 lines. Thetime and concentration dependence of the effects on cholesterolaccumulation in the LSOs were tested. For the most effective polymers, adetermination was made on how long they are retained in cells. In orderto quantify cellular uptake and retention of the polymers, the initialtest polymers also included fluorophores. The retention ability wasfound by measuring the loss of fluorescence of the polymers whenincubated without polymer in the surrounding medium. In parallel, adetermination was made on how long the reduction of cholesterolaccumulation persists after removal of the polymer from the growthmedium.

In this experiment, studies were conducted to determine the effects ofMβCD on NPC1 and NPC2 cells. FIG. 1 shows the concentration and timedependence of the removal of cholesterol from LSOs as assayed by apreviously described method based on filipin staining and digital imageanalysis (N. H. Pipalia, et al., J. Lipid Res., 47 (2006) pp. 284-301,and Rosenbaum A. I., et al. Biochim. Biophys. Acta., 1791 (2009), pp.1155-1165). From FIG. 1, it can be seen that the effect reaches steadystate in about 2 days, and 2-3 μM gives a half-maximal effect.Significantly, hydroxypropyl-βCD (i.e., “HPβCD”), a CD derivative thathas previously been used in animal studies, required about 3-5 timeshigher concentrations to achieve the same effect. To distinguish betweeneffects of extracellular MβCD and endocytosed MβCD, a brief (1 hour)incubation was conducted with a high concentration of MβCD (0.33 mM) toallow pinocytic uptake, which is well known to deliver soluble polarmolecules to late endosomes and lysosomes (S. Mukherjee, et al.,Physiol. Rev., 77 (1997) pp. 759-803). The cells were then rinsed andincubated in complete medium for 24 hours. During the chase, there was asignificant reduction in the cholesterol in LSOs, even with virtually noextracellular MβCD (FIG. 2). To further emphasize this, the one-hourloading pulse was conducted with cholesterol-loaded MβCD, which wouldactually increase the cellular cholesterol during the loading period.Nevertheless, there was a very significant loss of cholesterol from theLSOs during the subsequent 24 hr chase. These data provide strongevidence that endocytosed MβCD can fully account for the effects ofMβCD. Similar effects were seen on NPC2 cells (not shown).

Next, to test the idea of creating lysosomally targeted MβCD, dextranpolymer conjugates with a MβCD at the end of 15 atom polyethylene glycol(PEG) spacers were prepared. The original syntheses incorporated abouttwo MβCD per 70 KDa dextran chain; however, the number of MβCD can besignificantly increased, if desired. Even with this low degree ofsubstitution, the polymers were effective at lowering cholesterol in theLSOs (FIG. 3). Dextrans are delivered to late endosomes/lysosomes byfluid phase endocytosis (S. Mukherjee, et al., Physiol. Rev., 77 (1997)pp. 759-803).

Further studies showed that treatment with either MβCD or HPβCD reducedcholesterol accumulation, as detected by filipin labeling (FIG. 4). TheCD effects on LSO cholesterol accumulation in NPC1 and NPC2 mutant cellswere found to be dose and time dependent (FIG. 5). As shown, both MβCDand HPβCD can reduce cholesterol accumulation in LSOs of NPC1 and NPC2fibroblasts to near-normal levels using low micromolar concentrations. Acomparison of LSO values for untreated NPC1, NPC2, and apparently normalcells is shown in FIG. 6. Most of the decrease in LSO cholesteroloccurred during the first two days of incubation. MβCD is more potentthan HPβCD in eliciting this reduction (see Table 1 below). This findingcorrelates well with previous studies, which showed that MβCD is morepotent than HPβCD at extracting cholesterol from biological membranes.As shown by FIG. 7, studies employing gas chromatography and massspectrometry further demonstrate that overall cholesterol levels werereduced significantly upon CD treatment.

TABLE 1 Apparent EC₅₀ values for MβCD and HPβCD effects on LSO values ofGM03123 and GM18445 cells as function of time of incubation with thecompounds. Cell line Compound Days of treatment EC₅₀ (μM) R² GM03123MβCD 1 2.22 0.943 GM03123 MβCD 2 1.10 0.997 GM03123 MβCD 3 0.98 0.995GM03123 MβCD 4 1.08 0.993 GM18455 MβCD 1 40.39 0.987 GM18455 MβCD 2 3.190.965 GM18455 MβCD 3 2.80 0.999 GM18455 MβCD 4 1.69 0.997 GM03123 HPβCD1 3.16 0.781 GM03123 HPβCD 2 3.77 0.920 GM03123 HPβCD 3 4.18 0.966GM03123 HPβCD 4 5.44 0.991 GM18455 HPβCD 1 272.41 0.970 GM18455 HPβCD 220.84 0.909 GM18455 HPβCD 3 16.94 0.998 GM18455 HPβCD 4 11.51 0.991Apparent EC₅₀s (compound concentration at 50% reduction in LSO filipin)for MβCD and HPβCD treatment of GM03123 (NPC1) and GM18455 (NPC2) cellsfor 1-4 days were determined by fitting (using MATLAB, nonlinearleast-squares Levenberg-Marquardt algorithm) dose-response curves (FIG.5) to the rectangular hyperbola of the form y = m/(x + b) + c, andsolving for y = 50, where y = normalized LSO filipin values (%), x =compound concentration (μM), and m, b, and c are coefficients.

EXAMPLE 3 Effects of β-CDs After Treatment: Withdrawal Studies

To determine whether the CD-mediated decrease in cholesterolaccumulation would be sustained after the compound has been removed fromthe culture medium, NPC1 and NPC2 mutant cells were treated for I daywith varying concentrations of MβCD or HPβCD (FIG. 8). The cells werethen washed extensively to remove the compounds and returned to growthmedium for up to three days. Cells that were allowed to grow for onemore day after treatment with CD had a further decrease in LSO valueswhen compared with cells that were treated for the same length of timebut fixed immediately after treatment. After two and three days aftertreatment, cholesterol accumulation in LSOs of both NPC1 and NPC2 mutantcells started to increase, but there was still a significant reductionin the LSO cholesterol accumulation even after three days in serumcontaining medium in the absence of extracellular CD.

The delayed nature of CD effects (i.e., a further decrease incholesterol accumulation after CD is removed from the medium) providedan initial indication that CD is acting to transfer cholesterol withinthe LSOs, as opposed to eliciting net efflux from. LSOs by loweringcholesterol levels in the PM.

EXAMPLE 4 Acute MβCD Treatments of NPC-Defective Cells

To determine whether the reduction of free cholesterol in LSOs ofNPC-defective cells resulted from extraction of cholesterol by CD fromthe PM or from inside the LSOs, this experiment acutely treated cellsfor one hour with MβCD in medium without serum (to extract cholesterolfrom the PM), MβCD in medium plus 10% PBS (to exchange cholesterolbetween serum lipoproteins and the PM (V. M. Atger, et al. (1997), J.Clin. Invest., 99:773-780)), or MβCD loaded with cholesterol (≈5:1(MβCD/cholesterol) ratio) to overload cells with cholesterol.

Cells that were fixed immediately after treatment with MβCD (with orwithout serum) showed no marked decrease in LSO filipin staining (FIG.9). The cells that were treated with MβCD/cholesterol showed an overallincrease in filipin staining (FIGS. 10B and 10D). Quantitative analysisof images taken 24 hours after the treatment with CD revealed that,regardless of the method of initial treatment, there was a significantreduction in filipin staining of LSOs of treated cells as compared withuntreated controls (FIG. 10). These findings are consistent with amechanism in which CD is acting from the inside LY/LE to compensate forthe lack of functional NPC1 or NPC2.

EXAMPLE 5 MβCD-Dextran Conjugates also Reduce Cholesterol Accumulationin NPC Mutant Cells

The endocytic uptake of dextran polymers and their delivery to the LE/LYhas been characterized in many studies (S. Mukherjee, et al., Physiol.Rev., 77 (1997) pp. 759-803). To ensure CD delivery to LSOs, MβCD wascovalently conjugated to dextran polymers via a polyethylene glycollinker. As shown in FIG. 11A, MβCD-dextran can also reverse cholesterolaccumulation in the LSOs of NPC1-defective cells. To visualize CDtrafficking, MβCD-dextran conjugates were labeled with AlexaFluor546.This conjugate was observed to be localized to intracellular organellesthat were labeled with filipin (FIG. 11B). Thus, theMβCD-dextran-AlexaFluor546 polymer has been shown to be in the LSOs.

EXAMPLE 6 Effects of Acute CD Treatments on Cholesterol Esterification

To measure cholesterol levels within the endoplasmic reticulum inresponse to brief CD treatments, cholesterol esterification was measuredby ACAT (FIG. 12). Increased esterification of cholesterol was observedseveral hours after a short (1-hour) CD treatment of NPC1- orNPC2-deficient cell lines. This increase was inhibited by compoundS58-035, an ACAT inhibitor (A. C. Ross et al., (1984) J. Biol. Chem.259:815-819.), thus demonstrating that the increase in esterificationwas mediated by ACAT. Corresponding reductions in cholesterolaccumulation upon MβCD treatment, as measured by the LSO filipin assay,are shown in FIG. 13.

EXAMPLE 7 β-CD Effects on U18666A-Treated Fibroblasts

To test whether the apparent EC50 for clearance of cholesterol from LSOdepended on cholesterol levels accumulated and to examine this in anisogenic background, this experiment used apparently normal humanfibroblasts (GM05659) treated with various concentrations of the NPCphenotype-inducing compound U18666A(3-β-[(2-diethyl-amino)ethoxy]androst-5-en-17-one). U18666A is a classII amphipathic amine that has been reported to impair cholesterol effluxfrom LE/LY and create an NPC-like phenotype in treated cells (R. J.Cenedella, (2009) Lipids 44:477-487). The extent of cholesterolaccumulation in LSOs of U18666A-treated cells is dose dependent (FIG.14A). This accumulation can be reversed in a dose-dependent manner byusing either MβCD or HPβCD (FIGS. 14B and 14C). The EC50s for clearanceof cholesterol from U18666A-treated cells are summarized in Table 2(below), and the dependence of EC50 on U18666A concentration isdisplayed in FIG. 14D. Higher levels of cholesterol in the cells requirehigher concentration of CD for clearance. The increased potency of MβCDvs. HPβCD has thus been demonstrated once again, and it seems to be moreprominent for higher levels of cholesterol accumulation.

TABLE 2 Apparent EC₅₀ values for MβCD and HPβCD effects on LSO values ofU186664-treated GM05659 cells as function of U18666A concentration.Concentration U18666A (nM) 111 333 1,000 EC₅₀ MβCD (μM) 6.2 18.9 48.1EC₅₀ HPβCD (μM) 17.4 69.9 184.9 Apparent EC₅₀S (compound concentrationat 50% reduction in LSO filipin) for MβCD and HPβCD treatment of GM05659apparently normal fibroblasts treated with 1M U18666A. Cells werepretreated with 1 μM U18666A for 1 day and then treated with therespective cyclodextrin for another day, in the continued presence of 1μM U18666A compound. EC₅₀s were calculated as in Table 1. All fits hadR² ≧ 0.97.

EXAMPLE 8 Effects of β-CDs on BMP Accumulation in NPC-Defective Cells

Because levels of BMP are also elevated in the LSOs of NPC mutant cell(J. Chevallier, et al. (2008) J. Biol. Chem. 283:27871-27880), thisexperiment examined the effects of CDs on BMP accumulation in NPC mutantcells. Both MβCD and HPβCD significantly reduced BMP accumulation, asmeasured by quantification of images of cells stained with anti-BMPantibody (FIG. 15). This indicates that secondary lipid accumulation wasreduced along with cholesterol changes. Relative BMP levels in NPC1,NPC2, normal, and normal cells treated with U18666A are shown in FIG.16.

EXAMPLE 9 Discussion of Results

Studies in npc1−/− mice have demonstrated that single injections ofHPβCD can extend their lifespan (e.g., F. Camargo, et al. (2001) LifeSci. 70:131-142; B. Liu et al. (2008) J. Lipid Res. 49:663-669; B. Liu,et al. (2009) Proc. Natl. Acad. Sci. USA, 106::2377-2382; S.Lope-Piedrafita, et al. (2008) J. Neurosci. Res. 86:2802-2807.).Subsequent studies with repeated injections of HPβCD have shown furtherprolongation of lifespan as well as clearance of variousglycosphingolipids (C. D. Davidson, et al. (2009) PLoS One 4:e6951).These animal studies did not address the mechanism of action of CDs. Arecent study (L. Abi-Mosleh, (2009) Proc. Natl. Acad. Sci. USA106:19316-19321) used cultured NPC1- and NPC2-deficient cells to showthat HPβCD treatment increased ACAT-mediated esterification ofcholesterol even in the absence of functional NPC proteins.

In these experiments, the role of CD in reducing cholesterolaccumulation in NPC mutant cells has been examined. CDs are hydrophilic,membrane impermeant molecules that can reach the inside of LE/LY viapinocytosis, similar to other membrane-impermeant molecules (Mukherjee1997, Ibid.). Mammalian cells lack the enzymes for degradation of CDs(M. E. Davis et al., (2004) Nat. Rev. Drug. Discov. 3:1023-1035), andthus, CDs delivered to LE/LY are expected to remain intact.

Several lines of evidence suggest that the major effect of CDs inreducing cholesterol accumulation is attributable to endocytosed CD. Theeffects on cholesterol reduction in LSOs continue for several days afterextracellular CD has been removed from the medium (FIG. 8). The slowloss of effectiveness of the CD would be consistent with reducedconcentrations in the endocytic system as a consequence of cell divisionand vesicular transport out of lysosomes. It was also found that brief(1 hour) incubations with CD leads to a reduction in LSO cholesterolafter about 24 hours in the absence of extracellular CD. This wasobserved even when the initial incubation was with cholesterol-loadedCD, which caused an initial increase in cellular cholesterol levels(FIG. 10). In experiments where MβCD was conjugated to dextran polymersto ensure its delivery to the LSOs, it was found that this compound wasable to correct NPC1 deficiency (FIG. 11A). Additionally, it was foundthat these conjugates were delivered to LSOs (FIG. 11B). Theseobservations further demonstrate that CDs can act from inside the lumenof the LSO.

Both NPC1 and NPC2 mutant cells were tested in this study, and both celltypes responded well to treatment by CDs. These findings indicate thatCDs are capable of replacing the function of either NPC1 or NPC2.Because NPC2 is a small, soluble protein that has been shown to transfercholesterol between membranes in vitro (J. Storch, et al. (2009)Biochim. Biophys. Acta. 1791:671-678.), its functional replacement by CDcan be envisaged easily. The mechanism underlying the ability of a CD tobypass the requirement for functional NPC1 is less clear. NPC1 is alarge transmembrane protein residing in the limiting membrane of LE/LY,and its role in cholesterol efflux is not well understood despite recentadvances in understanding its cholesterol binding properties (H. J.Kwon, et al. (2009) Cell 137:1213-1224; R. E. Infante, et al. (2008)Proc. Natl. Acad. Sci. USA 105:15287-15292). It is possible that tofacilitate cholesterol egress from LE/LY, NPC2 has to interact with NPC1to deliver cholesterol to the limiting membrane for egress (L.Abi-Mosleh, (2009) Ibid.; H. J. Kwon, et al. (2009) Ibid.), whereas a CDcan bypass that requirement and deliver cholesterol directly to thelimiting membrane. Once in the limiting membrane, cholesterol couldleave the LE/LY by vesicular or nonvesicular transport processes (B.Mesmin, (2009) Biochim. Biophys. Acta. 1791:636-645) to be delivered toother organelles.

To verify that the cholesterol liberated by CD treatments reaches thecytosolic compartment, other experiments were conducted to measureesterification of cholesterol by ACAT. In those experiments, it wasfound that short treatments of either NPC1 or NPC2 cells with either CD,followed by a ˜46-hour chase, lead to an increase in cholesterolesterification by ACAT, thus indicating that more cholesterol isdelivered to the endoplasmic reticulum in CD-treated cells (FIG. 13).

BMP is an unusual lipid that is present in high levels in lateendosomes, and elevated levels of cholesterol associated with NPCmutations or treatment with U18666A lead to elevated levels of BMP inthe LSOs (J. Chevallier, et al. (2008) Ibid.; T. Kobayashi, et al.(1999) Nat. Cell Biol. 1:113-118). To verify that the effects of CDtreatment go beyond merely cholesterol reduction, BMP levels weremeasured in treated and untreated cells. From these experiments, it wasfound that the BMP levels were reduced significantly by CD treatments(FIG. 15). This is consistent with the observation in npc1−/− mice thatCD treatments reduce levels of storage of other lipids in addition tocholesterol (C. D. Davidson, et al. (2009), Ibid.).

The work described herein supports the utility of the instant CD-basedcompositions for the treatment of NPC disease. Furthermore, it has beenfound herein that MβCD produces effects equivalent to those of HPβCD atapproximately three-fold lower concentrations. This is consistent withthe better ability of MβCD to extract cholesterol from biologicmembranes. This observation may be important because relatively highamounts of HPβCD need to be injected to obtain a partial therapeuticeffect (e.g., F. Camargo, et al. (2001) Life Sci. 70:131-142; B. Liu etal. (2008) J. Lipid Res. 49:663-669; B. Liu, et al. (2009) Proc. Natl.Acad. Sci. USA, 106::2377-2382; S. Lope-Piedrafita, et al. (2008) J.Neurosci. Res. 86:2802-2807). Additionally, the observation that CDworks from inside LE/LY suggests that modifications that target CDs forendocytic uptake and retention might significantly enhance its potency.

While the main focus in these examples has been optimizing intracellulartargeting of MβCD, the methods described herein are also useful fortargeting compounds within the body. In particular, it has been reportedthat some peptides, such as a 29 residue peptide from the rabies viruscoat protein, can bind to receptors on brain endothelial cells andfacilitate transcytosis into the brain (P. Kumar, et al., Nature, 448(2007) pp. 39-43). It is relatively easy to link such peptides to thepolymeric compositions of the instant invention. Such peptides have beenfound to not interfere with the intracellular targeting and cholesterolreduction properties of the polymers.

EXAMPLE 10 HDAC Inhibitors

Tests were conducted to determine the ability of several HDAC inhibitors(HDACi) to reduce cholesterol in the LSOs following a 48-hour treatmentat various concentrations. The structures of six of the HDAC inhibitorsused in this study are shown in FIG. 17. The dose dependence of variousHDACi inhibitors after the 48-hour treatment is shown in FIGS. 18A and18B. FIG. 18A shows the results for NPC1 human fibroblast GM18453. FIG.18B shows the results for NPC2 human fibroblast GM18445. Each data pointin the plot is representative of a total of 32 images from twoindependent experiments. The standard deviation (SD) for DMSO is 0.07,error bar=SE. The dotted line represents the DMSO control. As shown,both trichostatin A (TSA) and LBH-589 were significantly effective at 37nM, and LBH-589 was effective at 4 nM.

Dose and time dependence for each of the studied HDACi compounds as afunction of time on NPC1 fibroblast GM03123 are shown in the plots ofFIG. 19. Each data point in the plot is representative of a total of 32images from two independent experiments. The SD for DMSO is 0.07, errorbar=SE. The dotted line represents the DMSO control. SAHA was somewhateffective at 1 μM, and it was much more effective in other experimentsat 10 μM (not shown). Both SAHA and LBH-589 target class I & II HDACs,and the relative potency in the cholesterol reduction parallels theirrelative potency for inhibition of HDAC (M. Dokmanovic, et al., Histonedeacetylase inhibitors: overview and perspectives, Mol. Cancer Res., 5(2007) pp. 981-989).

Some HDAC inhibitors were found to be remarkably effective in reducingcholesterol accumulation in LSOs. It should be noted that some of theseHDAC inhibitors are in use for treating neuroblastoma, so theyapparently cross the blood-brain barrier easily.

Several other HDAC inhibitors were assayed. Concentration dependence wasassayed as before, and assays were conducted at a range of exposuretimes from 4 hours to 4 days. The results show that two inhibitors ofthe Class I and II HDACs are very potent in reducing cholesterolaccumulation.

Effects of the HDACs on LDL uptake, cholesterol synthesis, lysosomalacid lipase activity, ACAT activity, and cholesterol efflux were alsocharacterized to identify which cellular processes are responsible forlowering accumulated cholesterol. HDACs have many cellular targets, andtheir effects can be pleiotropic. In addition to histones, in which theacetylation state can affect expression of many genes, many cytoplasmicproteins are also regulated in their function byacetylation/deacetylation. For some of the HDAC inhibitors mosteffective at reducing LSO cholesterol, a variety of routine biochemicalassays were employed to determine which step(s) of cholesterol transportor metabolism are being affected.

FIG. 20 shows the effect of various HDACi compounds on cellproliferation. The effect was monitored at 4 h, 24 h, 48 h and 72 h posttreatment by counting the number of nuclei. Each data point in a plot isrepresentative of a total of 32 images from two independent experiments.Error bar=SE.

FIG. 21 shows data from a filipin assay after HDAC silencing in GM03123cells by electroporation. The plot is representative of threeindependent experiments for HDAC 6 and two independent experiments forHDAC7A.

The results above demonstrate that HDAC inhibitor treatment dramaticallyreduces cholesterol accumulation in lysosomal storage organelles of NPC1skin fibroblasts.

EXAMPLE 11 Acid SMase Restoration

Materials and Methods

The tissue culture plasticware used in these experiments was purchasedfrom Fisher Scientific Co. Tissue culture media and other tissue culturereagents were purchased from Invitrogen Corp. FBS was obtained fromGemini Bio-Products. LDL (d, 1.020-1.063 g/mL) from fresh human plasmawas isolated by preparative ultracentrifugation as described elsewhere(Havel 1955). Radiochemicals were purchased from either Perkin-ElmerLife and Analytical Sciences, Inc., or American Radiolabeled Chemicals,Inc. All restriction enzymes, Antarctic phosphatase and T4 DNA ligasewere purchased from New England BioLabs. Recombinant human acid SMasewas prepared from transfected CHO cells and purified as previouslydescribed (He 1999). All other chemicals and reagents were fromSigma-Aldrich, and all organic solvents were from Fisher Scientific Co.

Cells. CHO, 25RA (Chang 1980) and CT60 cells (Cadigan 1990; Watari 1999)were grown in monolayer cultures in Ham's F12 medium containing 10% FBS.Human WT (GM05659) and NPC (GM03123) Fbs, from Conic11 Institute ofMedical Research, were cultured in Modified Eagle's Medium (MEM)containing 10% FBS. The NPC Fbs were derived originally from a9-year-old compound heterozygote female, with missense mutations in exon6 of one allele (P237S) and exon 21 of the other allele (I1061T). Thefibroblasts from this subject expressed no detectable NPC1 protein byimmunoblot analysis and, as expected, had a severe defect in thetrafficking lipoprotein-derived cholesterol (Yamamoto 2000; Pentchev1985). The GM18453 NPC Fbs were derived from a male donor with ahomozygous mutation at I1061 T. All cells were plated at a minimum of 24h prior to commencement of the experiment.

Acid SMase activity assay. Cell extracts were prepared by scraping cellsinto ice-cold 250 mM sucrose, followed by sonication on ice twice for 10seconds each using a Branson Sonifier 450. To prepare the substrate,solvent from 0.1 μCi [cholinemethyl-¹⁴C] SM (52 mCi/mmol; Perkin-ElmerLife and Analytical Sciences) was evaporated, and the labeled SM wasresuspended in 20 μL of assay buffer (100 mM sodium acetate pH 5.0, 100μM ZnCl2) containing 2.7% Triton X-100 and vortexed for 2 minutes. Theassay solution, which was added to 1.5-mL microcentrifuge tubes on ice,consisted of 50 μL assay buffer, 20 μL substrate and 20 μL of cellextract. After incubation for 60 minutes at 37C, the reaction wasterminated by adding 125 μL of chloroform:methanol (2:1, v/v). The assaytubes were vortexed for 1 minute and then centrifuged 5000 g for 5minutes at 4° C. A 50-μL aliquot of the upper aqueous phase was removedfor scintillation counting to determine the amount of [¹⁴C]phosphorylcholine released from [¹⁴C]SM. The protein content of the cellextracts was assayed using the method of Lowry (Lowry 1951).

Cellular cholesterol mass determination by gas chromatography. In Ham'sF12 growth medium supplemented with 10% FBS, 25RA, CT60, CT60-VEC,CT60-WT and CT60-C629 cells were grown. Lipids were extracted from thecells with hexane:2-propanol (3:2) and separated on a Varian Factor Fourcapillary column (VF-1 ms 30 m 0.25 m in IDDF 0.25) using Varian 4000GC/MS/MS system. The injector temperature was 270° C. The followingtemperature gradient was used: initial temperature was 115° C., whichwas raised to 260° C. at 9° C./min and held for 2.89 minutes, thenraised to 269° C. at 3° C./min and again to 290° C. at 9° C./min andheld for 4.67 minutes. Flow rate was 5 mL/min He(g). Electron ionizationwas used with the current set at 10 μA. Total ionic current was used fordetection (50-1000 m/z). β-Sitosterol was used as an internal standardfor quantification of μg free cholesterol per mg cell protein. Proteinconcentration was determined with modified Lowry reagent (Bio-Rad).

Sphingomyelin mass assay. Total cellular SM mass was quantified in lipidextracts of cells using the TLC-Bartlett procedure as previouslydescribed (Okwu 1994; Bartlett 1959), except that the scraped thin-layerchromatography (TLC) spot was extracted with methanol:chloroform (2:1)

Preparation of WT or C629S SMPD1 constructs. A plasmid containing thehuman cDNA for acid SMase (SMPD1)(56) was digested with EcoRI to releasethe cDNA. The cDNA was then ligated to phosphatase-treated,EcoRI-digested pBSIISK (Stratagene) to generate pBS.SMPD1. To obtain the3 ends of the cDNA designed to encode WT or C629S acid SMase, polymerasechain reaction (PCR) was conducted using the high-fidelity Platinum PfxDNA polymerase (Invitrogen) and the pBS.SMPD1 plasmid as the template.For both constructs, a NotI site was created after the stop codon toassist in the cloning process. The primers for the WT constructconsisted of the sense primer SMPD11469 (5-ACTGTCTGAAGAGCTGGAGCT-3) andthe antisense primer 5TTTTATTGCGGCCGCCTAGCAAAACAGTGGCCTTGG-3. The senseprimer for the C629S construct was SMPD11469 and the antisense primerwas 5-TTTTATTGCGGCCGCCTAGGAAAACAGTGGCCTTGG-3 (the codon for serine inposition 629 is underlined). Each PCR product was digested with SphI(which cuts at position 1860 of SMPD1 cDNA) and NotI, and thenseparately ligated to the 4.8-kb SphI-NotI fragment from pBS.SMPD1 togenerate pBS.WT or pBS.C629S, in which the polyadenylation sequence wasremoved from the SMPD1 cDNA. The final vectors were created in theexpression vector pIRES-hrGFP II (Stratagem) by ligating the 6-kbEcoRI-NotI fragment from the vector with the 2-kb EcoRI-NotI fragmentfrom either pBS.WT or pBS.C629S to generate pIRES.WT or pIRES.C629S. TheDNA sequence was confirmed for each construct. The vector pIREShrGFP IIconsists of the SV40 promoter driving the expression of theneomycin-resistance gene and a bicistronic expression cassette under thecontrol of the human cytomegalovirus promoter which contains a multiplecloning site followed by an internal ribosome entry site (IBES) that islinked to the GFP coding sequence.

Transfection of cells with the WT or C629S SMPD1 constructs. Fortransient transfection, CT60 cells in 16-mm wells were transfected with340 ng empty vector (pIRES-hrGFP II; VEC), pIRES.WT or pIRES.C629S usingLipofectamine 2000. For stable transfection, CT60 cells were transfectedwith the empty vector, pIRES.WT or pIRES.C629S, and then selected forgrowth in G418 (0.5 mg/mL) and named, respectively, CT60-VEC, CT60-WTand CT60-C629S. Human fibroblasts were transiently transfected using areverse format with Effectene (Qiagen, Valencia, Calif.) as thetransfection reagent. Briefly, VEC, WT and C629S vectors (above) weremixed with Effectene transfection reagent, enhancer andmanufacturer-supplied buffer (EC) in the ratio recommended by themanufacturer and dispensed in different wells of a six-well plate. Thefibroblasts were then added to the above mixture in suspension andincubated overnight at 37° C. in a tissue culture incubator. Freshgrowth medium was added to the cells and, after an additional 6 h ofincubation, the cells were fixed and analyzed for filipin and/or GFPstaining (as described below).

Acid SMase enzyme replacement. Normal (GM05659) and NPC1 humanfibroblasts (GM03123) were incubated in a medium containing 3 μg/mLrecombinant human acid SMase (rhASM). Two days later, the cells werewashed thoroughly with PBS, and either lysed and assayed for acid SMaseactivity or fixed and stained with filipin for imaging andquantification. NPC1 human fibroblasts GM18453 were treated with 3 μg/mLrhASM for 24 h before fixing, staining with filipin, imaging andquantifying.

Alexa555-conjugated rhASM enzyme replacement. For identifying thelocalization of rhASM in the cells, recombinant enzyme was conjugatedwith Alexa555 (Invitrogen) according to the manufacturer's protocol.Normal (GM05659) and NPC1 human fibroblasts (GM03123) were incubated ina medium containing 3 μg/mL Alexa555-rhASM for 24 h prior to fixing andstaining with filipin. Images were acquired using standard UV and TRITCfilters for filipin and Alexa555-labeled rhASM, and quantified.

Filipin staining. Cells were plated onto poly-d-lysine-coated 35-mmcoverslip dishes in a medium containing 10% FBS. After 1-2 days ofgrowth, the cell monolayers were washed three times with PBS and thenfixed with 3% paraformaldehyde in PBS for 20 minutes at roomtemperature, followed by three more washes with PBS. To detect freecholesterol, filipin was added to the fixed cells (50 μg/mL in PBS) for45 minutes at room temperature. Finally, the cells were washed threetimes with PBS, and images were acquired immediately after labeling.

Fluorescence microscopy. Fluorescence microscopy and digital imageacquisition were carried out using a Leica DMIRB microscope (LeicaMikroscopie und Systeme GmbH) equipped with a cooled Charge CoupledDevice (CCD) camera (Princeton Instruments) and driven by MetaMorphImaging System acquisition software (MDS Analytical Technologies). Allimages were acquired using an oil immersion objective (63×, 1.25 NA).Filipin was imaged using an A4 filter cube obtained from ChromaTechnology Corp.: 360-nm (40-nm bandpass) excitation filter; 365 DCLP(DiChroic Long Pass) filter and 480-nm (40-nm bandpass) emission filter.To minimize photo-bleaching of the filipin signal, a neutral densityfilter transmitting 1.5% light was used to acquire images. GFP wasimaged using a standard FITC filter cube. Fluorescence crossover fromone channel to another was measured using single-labeled samples of eachprobe and was found to be insignificant.

Image analysis. Images were analyzed using Metamorph (version 7.0 r4)image analysis software from Molecular Devices (MDS AnalyticalTechnologies). All images were corrected for background before analysis.The average filipin and LSO ratio was calculated as described previously(Pipalia 2006). The LSO ratio was calculated based on low-thresholdedregion and high-thresholded intensities in the filipin image asdescribed previously (Pipalia 2006).

Cholesterol efflux assay. [³H]cholesteryl ester (CE)-labeled LDL wasprepared as described by Krieger, 1986. Briefly, the lipid core of LDLwas replaced with [1,2,6,7-³H(N)] cholesteryl oleate (AmericanRadiolabeled Chemicals, Inc.). The specific activity of the labeled LDLwas 13.7 cpm/ng of protein. Cells were plated in 24-well plates andincubated for 2 days in F12 medium containing 10% lipoprotein-deficientserum. The cells were labeled by incubation for 4 h in F12 mediumcontaining 0.2% BSA and 10 μg/mL [³H]CE-labeled LDL. At the end of thispulse period, the cells were washed and the medium was replaced withF12/BSA medium containing 50 μg/mL HDL3. At the indicated time points,100 μL of media was removed and centrifuged for 5 minutes at 14000×g toremove cellular debris, and the radioactivity in this portion of themedia was determined by liquid scintillation counting. After the lasttime point, the cells were washed and the monolayer was dissolved in 250μL of 0.1N NaOH at room temperature for a minimum of 4 hours. A 100-μLaliquot of the cell lysate was measured, and the percent efflux wascalculated as [(media cpm)/(cell+media cpm)]×100. To obtain the valuefor HDL3-stimulated efflux, the percent efflux in the absence of HDL3was subtracted from the percent efflux in the presence of acceptor.

Transferrin efflux kinetics as a measure of TfR recycling. CT60 cellsexpressing the human TfR (CT60hTfR cells) (Pipalia 2007) were stablytransfected with the empty vector pIRES-hrGFPII, pIRES.WT or pIRES.C629Sand sorted for high levels of green fluorescence. The cells weremaintained in a medium containing G418 (0.5 mg/mL). The resulting stablecell lines were named, respectively, CT60hTfR-VEC, CT60hTfR-WT andCT60hTfR-C629S. The recycling of TfR from endosomes to cell surface wasmeasured as previously described elsewhere (Johnson 2001). Briefly, thecells were plated in 12-well culture dishes in bicarbonate-bufferedMcCoy's medium. For each experiment and for each cell type, six plateswere prepared for six time points (3, 5, 10, 15, 30 and 60 min). Fourwells in each plate were pulsed with 5 μg/mL [¹²⁵I]-labeled Tf, and theremaining two wells were pulsed with 5 μg/mL [¹²⁵I]-labeled Tf, with a200-fold excess of unlabeled Tf to ascertain non-specific binding. Allplates were incubated for 30 minutes and washed once with serum-freeMcCoy's medium, once with acid wash buffer (pH 5.0), and twice withefflux medium. Finally, the efflux medium was added to each plate andincubated for varying time periods. At the end of each chase time, theefflux medium was transferred to collection tubes, and the cells werequickly washed once with the same medium, which was then added to thecollection tube. Solubilization solution was added to each well,triturated and transferred to another collection tube. The cells werewashed with water, and the washings were added to the solubilizationmedium. The amount of [¹²⁵I]-labeled Tf was measured for all the effluxand cellular fractions, and the recycling rate of TfR was determined.

BMP labeling. Cells seeded in poly-d-lysine-coated coverslip dishes andgrown for 2 days were fixed with 3% paraformaldehyde in PBS for 20minutes at room temperature. Fixed cells were subsequently permeabilizedwith 0.5% saponin (to ensure complete immunolabeling of BMP withinmultivesicular lysosomal compartments) and incubated with primary murineanti-BMP antibody (Echelon Biosciences Inc.) for 45 minutes. The cellmonolayers were washed three times with PBS and then incubated withAlexa546-labeled goat anti-mouse IgG for 1 hour in the presence of 0.1%saponin. Finally, the cells were washed three times with PBS. Imageswere acquired using wide-field epifluorescence microscopy at 63magnification and standard TRITC filters.

Assay for uptake of LDL-derived cholesterol. Cells were incubated for 2hours with 5 μg/mL [¹⁴C]CE-labeled LDL, which was produced using thesame method described above for [³]CE-labeled LDL. Lipids were extractedin hexane:isopropanol (3:2) and subjected to thin-layer chromatographyto separate free cholesterol and CE.

Statistics. Data are presented as mean ±SEM of triplicate experiments.Statistical significance was determined using the Student's t-test withunequal variance or one-way ANOVA and the Tukey's multiple comparisontest using GraphPad Prism version 4.03 for Windows (GraphPad Software)

Experiments in Acid SMase Restoration

Different primary lysosomal trafficking defects lead to commonalterations in lipid trafficking, suggesting cooperative interactionsamong lysosomal lipids. However, cellular analysis of the functionalconsequences of this phenomenon is lacking. As a test case, thisexperiment studied cells with defective Niemann-Pick C1 (NPC1) protein,a cholesterol trafficking protein whose defect gives rise to lysosomalaccumulation of cholesterol and other lipids, leading to NPC disease.NPC1 cells also develop a secondary defect in acid sphingomyelinase(SMase) activity despite a normal acid SMase gene (SMPD1). When acidSMase activity was restored to normal levels in NPC1 deficient CHO cellsthrough SMPD1 transfection, there was a dramatic reduction in lysosomalcholesterol. Two other defects, excess lysosomalbis-(monoacylglycerol)phosphate (BMP) and defective transferrin receptor(TfR) recycling, were also markedly improved. To test its relevance inhuman cells, the acid SMase activity defect in fibroblasts from NPC1patients was corrected by SMPD1 transfection or acid SMase enzymereplacement. Both treatments resulted in a dramatic reduction inlysosomal cholesterol. These data show that correcting one aspect of acomplex lysosomal lipid storage disease can reduce the cellularconsequences even if the primary genetic defect is not corrected.

Lysosomal lipid storage diseases (LSDs) are caused by mutations inspecific lysosomal hydrolases, trafficking proteins or their co-factors,leading to the accumulation of substrate compounds in lateendosome-derived structures called lysosomal storage organelles (LSOs).The primary cellular abnormality often perturbs the trafficking ofmultiple lipids and proteins, which probably contributes to the overallpathophysiology of the disease. These trafficking abnormalities andother secondary defects may, in turn, amplify the cellularpathophysiology triggered by the primary mutation. Identification andfunctional assessment of these secondary defects may therefore offer newtherapeutic opportunities even if the primary genetic defect is notcorrected.

To test this concept, this experiment studied cells lacking the lateendosomal protein Niemann-Pick C1 (NPC1). NPC1, a membrane protein,co-operates in some manner with NPC2, a cholesterol-binding protein inthe lumen of endosomes, to transfer endocytosed cholesterol from thelumen to the membrane of late endosomes. The cholesterol is thentransferred from the endosomal membrane to peripheral cellular sitesthrough one or more processes that are still under intenseinvestigation. Cells lacking functional NPC1 and NPC2 show defects inthe transport of cholesterol and other lipids from late endosomes toperipheral sites in the cell. Mutations in NPC1 and NPC2 give rise toNiemann-Pick C disease, which is characterized by hepatosplenomegaly,liver disease and potentially devastating neurological disease.

At the cellular level, cells with defective NPC1 accumulate cholesterolalong with excess sphingomyelin (SM), glycosphingolipids andbis-(monoacylglycerol)phosphate (BMP). It remains unclear whether theprimary defect in NPC1 mutants is directly associated with cholesteroltransport or whether the cholesterol accumulation is secondary toaccumulation of other lipids, which associate with cholesterol inmembranes. In this regard, it is noteworthy that cholesterol-enrichedNPC cells and tissues from NPC1-mutant mice and humans have a secondary,post-translational defect in the activity of a lysosomal enzyme, acidsphingomyelinase (SMase). This alteration in acid SMase activity can beobserved in wild-type (WT) cells with increased levels of late endosomalcholesterol resulting from incubation with low-density lipoprotein (LDL)and progesterone. Thus, elevated cholesterol and elevated SM appear tobe synergistically linked in a positive feedback loop.

Although the mechanism of suppression of acid SMase activity bycholesterol is not known, it can be surmised that this defect might havefunctional significance. In particular, primary deficiency of acid SMase(types A and B Niemann-Pick disease) leads to cellular defects anddisease characteristics that share certain features with NPC disease.For example, both types A/B and type C Niemann-Pick disease patients areknown to have hepatosplenomegaly and neurological abnormalities, and ithas previously been shown that macrophages lacking acid SMase havedefective late endosomal cholesterol trafficking (A. R. Leventhal, etal., J. Biol. Chem., 2001, 276:44976-44983). This study, therefore,aimed to better understand whether some of the trafficking defects incells with NPC disease could be corrected by correcting the secondaryenzymatic activity defect (i.e. acid SMase). The cell culture datapresented in this study certainly support this initially formulatedconcept.

Restoration of Acid SMase Activity in NPC1-Defective CT60 Cells byTransfection with WT and/or C629S Acid SMase (SMPD1) cDNA

As a model of NPC1 cells, this experiment studied CT60 cells, which areNPC1-deficient CHO cells derived from a parental line called 25RA(Cadigan 1990; Watari 1999). CT60 cells were observed to exhibit muchlower acid SMase activity than the 25RA cells (FIG. 22A) despite nodecrease in acid SMase protein. In preparation for studies addressingthe role of defective acid SMase activity in CT60 cells, this experimentsought to restore enzymatic activity in these cells through human acidSMase gene (SMPD1) transfection. For this purpose, this experiment usedtwo cDNA constructs: WT SMPD1 cDNA and a site-directed mutant cDNA(C629S SMPD1), which encodes acid SMase in which C-terminal Cys-629 ofthe enzyme is replaced with Ser. Qiu et al. (Qiu 2003) proposed thatC629S acid SMase mimics a naturally occurring processed form of theenzyme that has increased enzymatic activity.

As shown in FIG. 22A, cells transfected with either form of the cDNAdemonstrated restoration of acid SMase activity to a level similar tothat in the parental 25RA cells, whereas those transfected with aconstruct not containing the SMPD1 cDNA (VEC) had a level of acid SMaseactivity similar to that in non-transfected CT60 cells. Despite thisinitial hypothesis on the potential functional consequences of acidSMase in NPC1 cells, it was predicted that the overall quantitativeeffect of the NPC1 mutation and subsequent acid SMase correction ontotal cellular SM mass would be relatively modest, because only the poolof SM in late endosomes/lysosomes should be accessible to this enzyme.The observation was made that total cellular SM mass was 20% higher inCT60 and CT60 cells transfected with empty vector (CT60-VEC) comparedwith that in 25RA cells (p<0.05), and transfection of CT60 cells with WTor C6295 SMPD1 cDNA lowered SM mass close to the value in 25RA cells(data not shown). In summary, it was found that WT levels of acid SMaseactivity can be functionally restored in CT60 cells through transfectionwith either WT or C629S SMPD1 cDNA, thus allowing an assessment ofwhether this restoration can correct trafficking defects in these cells.

Restoration of Acid SMase Activity in CT60 Cells Leads to a Decrease inCholesterol Accumulation in LSOs

Previous mechanistic studies have shown that enrichment of membraneswith SM can disturb cholesterol trafficking because of SM-cholesterolinteractions, perturbations in membrane biophysical properties anddefective interaction of cholesterol transport proteins with SM-richmembranes (Cheruku 2006; Levanthal 2001; Ridgway 2000; Megha 2004). Thisexperiment, therefore, considered whether it would be possible tocorrect some of the trafficking defects in NPC cells by restoring normallevels of acid SMase activity. To test this hypothesis, the experimentquantified LSO filipin fluorescence in CT60 cells stably transfectedwith the cDNA constructs, as described in Materials and Methods. Shownin FIGS. 22B (a-c) is the expected increase in LSO filipin fluorescencein CT60 or CT60-VEC cells compared with that in 25RA cells.

Most importantly, the data show a striking loss of LSO filipinfluorescence in the cells expressing WT or C629S acid SMase (FIG. 22B, dand e therein). Quantification of LSO and whole-cell fluorescenceconfirmed these observations (FIGS. 22C,D). It has previously been shownthat estimation of cholesterol levels using filipin assay is comparablewith free cholesterol levels quantified by biochemical methods (Qin2006; Bartz 2009). To confirm these filipin results, this experimentalso estimated the free cholesterol using gas chromatography (FIG. 22E).A previous report showed that the free cholesterol in control 25RA cellsis only ˜40% compared with that of CT60 cells (Maguire 2005). Thisexperiment obtained similar data, as shown by the ˜45-50% decrease infree cholesterol content in control 25RA cells compared with that inCT60 or CT60-VEC cells. In agreement with these whole-cell filipinresults, it was found that the level of free cholesterol was markedlydecreased in CT60-WT and CT60-C629S cells and approached the level foundin 25RA cells. It is noted that the loss of LSO filipin fluorescence inthe SMPD I -transfected cells could not be explained by a decreaseduptake of LDL-cholesterol, which was similar among all five cell types(FIG. 22E). Thus, correcting the acid SMase activity defect inNPC1-mutant CT60 cells has a dramatic effect on improving a fundamentalcharacteristic of these cells, namely, cholesterol accumulation in LSOs.

Cholesterol leaving LSOs in acid SMase-transfected CT60 cells mightaccumulate in other cellular membranes, become esterified byacyl-CoA:cholesterol acyltransferase and/or get effluxed from the cells.The fact that whole-cell filipin fluorescence is lower in theSMPD1-transfected cells and becomes comparable with that in the parental25RA cells suggests that accumulation of high concentrations ofunesterified cholesterol in non-LSO sites is not a major fate of thecholesterol.

Moreover, when cells were incubated with [³H]cholesterol-labeled LDL for4 hours and then chased for up to 24 hours, an increase inesterification was not observed in CT60 cells transfected with SMPD1versus CT60-VEC cells (data not shown), thus suggesting that traffickingto and esterification by ACAT in the endoplasmic reticulum (ER) are alsonot a major fate of the acid SMase-mediated released LSO-derivedcholesterol. However, efflux of the LDL-derived [³H]cholesterol, whichis markedly decreased in CT60 cells, was restored by ˜20% after 24 h inSMPD1-transfected cells (FIG. 23A). The corresponding acid SMaseactivity measurements are shown in FIG. 23B. These data suggest that aportion of the cholesterol exiting the LSO in acid SMase-restored CT60cells is effluxed, with the rest probably being distributed diffusely toother cellular membranes.

Restoration of Acid SMase Activity in CT60 Cells Leads to a Decrease inBMP Accumulation and an Increase in the Half-Life of TransferrinReceptor (TfR) Recycling

The hydrophobic, acidic phospholipid, BMP, also calledlysobisphosphatidic acid (LBPA), has been shown to accumulate withinNPC1 cells (Pipalia 2007; Salvioli 2004). To test the possibility thatrestoration of acid SMase activity could have a broad corrective effecton NPC cells, this experiment investigated whether BMP accumulation wasalso diminished in SMPD1-transfected CT60 cells. Consistent with thedata cited above, CT60 cells accumulated much more BMP than 25RA cells(FIG. 24A, a-b therein). Cells transfected with non-SMPD1-containingcontrol vector also accumulated large amounts of BMP (FIG. 24A, ctherein). In contrast, cells transfected with WT or C629S SMPD1 hadgreatly diminished accumulation of BMP (FIG. 24A, d-e therein). Thequantified data are shown in FIG. 24B. Thus, it has been shown thatdefective BMP accumulation, like defective cholesterol accumulation, canbe partially corrected by restoration of acid SMase activity inNPC1-deficient cells.

It was previously reported that the recycling rates of TfRs aredecreased in NPC1 cells compared with that in normal fibroblasts(Pipalia 2007; Choudhury 2004). Using 25RA and CT60 cells that expressthe human transferrin receptor (hTfR) (Pipalia 2007), this experimentassessed TfR cycling in the control and SMPD1 transfected cells anddetermined whether the decrease in recycling in NPC1-deficient cellscould be corrected by restoration of acid SMase activity. To assay TfRrecycling, the cells were incubated with [¹²⁵I]Tf to achievesteady-state occupancy of the TfR with Tf. The Tf bound to the cellsurface was then removed, and the release of internal Tf from the cellswas monitored as a function of time. The [¹²⁵I]Tf released into themedium reflects the return of TfR from endosomes to plasma membrane. Thefraction of [¹²⁵I]Tf remaining in the cells decreases as a function oftime as a first-order process, and therefore the decrease incell-associated [¹²⁵I]Tf fits an exponential decay curve. It waspreviously reported that the Tf efflux kinetics yielded a t_(1/2) of11.3 minutes for 25RA cells and 21.7 min for CT60 cells (Pipalia 2007),which is consistent with the instant current results (FIG. 25). Asexpected, the TfR recycling defect persisted in CT60-VEC cells. Incontrast, cells expressing either WT or C629S acid SMase displayed amarked improvement in Tf trafficking, showing a t_(1/2) value close tothat of 25RA cells. Thus, it has been shown that the defect in TfRrecycling in CT60 cells, similar to the defect in LSO cholesterol andBMP accumulation, can be substantially corrected by restoration of acidSMase activity.

Genetic Restoration of Acid SMase Activity in Human NPC1 Cells Leads toa Dramatic Decrease in Cholesterol Accumulation in LSOs

To the extent that the secondary defect in acid SMase activity observedin NPC1 cells and in tissues from NPC mice and humans contributes to oneor more aspects of NPC disease pathology (see Discussion), the datashown herein raise the possibility that correction of this enzymeactivity defect may be clinically beneficial. Moreover, becauselysosomal pH is the same in control and NPC1 fibroblasts (NPC Fbs)(Lloyd-Evans 2008), the loss of acid SMase activity was not due toabnormal lysosomal pH. To explore this concept at the cellular level,this experiment conducted a series of acid SMase restoration experimentsusing skin fibroblasts isolated from a compound heterozygous child withthe late infantile form of NPC1 disease. NPC1 expression in these cellsis undetectable by immunoblot analysis, and the cells display a severedefect in trafficking lipoprotein-derived cholesterol (Yamamoto 2000;Pentchev 1985). As described previously (Maziere 1982), and verified inthis experiment (below), these fibroblasts also have a partial defect inacid SMase activity. Filipin staining of the cells revealed, asexpected, intense LSO staining, indicative of LSO cholesterolaccumulation (FIG. 26A, a-f therein, and first two bars (from left) ofplot shown in FIG. 26B). Cells transfected with the empty vector, whichcontained a GFP expression construct, showed marked filipin stainingthat was indistinguishable from untransfected cells (FIG. 26A, g-itherein, and third bar of plot shown in FIG. 26B). When the cells weretransfected with either the WT or the C629S SMPD1 cDNA construct, LSOfilipin staining was dramatically reduced in cells that weresuccessfully transfected (green) but not in cells that were notsuccessfully transfected (non-green), which acted as an internalnegative control (FIG. 26A, j-o therein, and last two bars of plot shownin FIG. 26B).

Restoration of Acid SMase Activity in Human NPC1 Cells by Exogenous AcidSMase also Leads to a Decrease in Cholesterol Accumulation in LSOs

Lysosomal enzyme defects, unlike defects in lysosomal membrane proteinssuch as NPC1, can be corrected both in vitro and in vivo using enzymereplacement therapy (Neufeld 1980; Brady 2006). Indeed, this approach iscurrently being tested in humans with primary acid SMase deficiency(Schuchman 2007). This strategy takes advantage of the fact that cellscan endocytose lysosomal enzymes and deliver them in a functionallyactive state to late endosomes and lysosomes (Neufeld 1980). To applythis concept to human NPC Fbs, the cells were pre-treated in the absenceor presence of recombinant human acid SMase (rhASM) and then assayed foracid SMase activity and LSO fluorescence after filipin staining. Asshown in FIG. 27A, the defect in acid SMase activity in the NPC Fbs wascorrected by pre-treatment with rhASM. Most importantly, acid SMasereplacement dramatically decreased LSO filipin fluorescence (FIG. 27B).Similar results were obtained using NPC1 skin fibroblasts from adifferent donor (GM18453) (FIG. 27C). Thus, restoration of acid SMaseactivity in NPC1 cells using two independent methods, genetic and enzymereplacement, has been shown to markedly correct the defect in LSOcholesterol accumulation.

Sub-Cellular Localization of Exogenously Added rhASM

To ascertain whether exogenously added rhASM was localized and processedin late endosomal organelles, WT (GM05659) and NPC1 (GM03123) humanfibroblasts were incubated for 24 hours in the absence or presence of 3μg/mL of Alexa555-labeled rhASM. To remove surface labeling, the cellswere further incubated for 15 minutes in a medium without enzyme. Thecells were then washed, fixed and stained with filipin for imaging andquantification. The uptake of rhASM-Alexa555 was completely blocked whenthe enzyme was added in the presence of excess mannose-6-phosphate (10mM) (data not shown). Conjugation of Alexa555 to the enzyme did notaffect its activity (data not shown). Shown in FIG. 28 are filipinimages (A, D and G) and Alexa555 images (B, E and H) for WT, NPC1 andNPC1+rhASM-Alexa555, respectively. Color overlays for filipin (green)and rhASM-Alexa555 (red) are shown in panels C, F and I of FIG. 28. Theimages in the inset are the zoomed color overlays of the regions markedin FIGS. 28C, F, and I. Exogenously added rhASM-Alexa555, presumablyinternalized via the mannose-6-phosphate receptor, specificallylocalized to cholesterol-laden storage organelles (as shown in FIGS. 28Cand I) and was not visible in the plasma membrane or other cellularorganelles. Quantification of LSO filipin and Alexa555-rhASM intensityafter incubation with 0 or 3 μg/mL Alexa555 conjugated rhASM for 24hours in WT and NPC (GM03123) Fbs (±SEM) is shown in FIGS. 28J and K.Note that the higher level of uptake of the rhASM in the NPC Fbs may beat least partly explained by the previous finding that expression ofIGF2/MPR is increased in human NPC fibroblast (Kobayashi 1999).

Overcoming Secondary Inactivation of Acid SMase

To validate the hypothesis that loss of acid SMase activity is asecondary effect of cholesterol storage in NPC1 patients, thisexperiment treated normal (GM05649) and NPC1 (GM03123) fibroblasts with0, 0.2 and 1.8 μg/mL rhASM. Acid SMase activity was assayed in parallelwith LSO filipin quantification as shown in FIGS. 29A,B. The acid SMaseactivity in wild-type fibroblasts (WT Fbs) (GM05659) increased slightlywith 0.2 μg/mL rhASM (p<0.05), and the activity did not increase furtherat 1.8 μg/mL (black bars in FIG. 29A). The minimal amount of LSO filipinstaining evident in normal fibroblasts (GM05659) remained low andconstant for all doses of rhASM tested (black bars in FIG. 29B). Incontrast, the addition of 0.2 μg/mL rhASM to NPC Fbs (GM03123) increasedacid SMase activity significantly with a corresponding modest decreasein LSO filipin intensity (˜20%), which indicates a decrease in LSOcholesterol accumulation (hashed bars in FIGS. 29A,B). Most importantly,increasing the rhASM dose to1.8 μg/mL resulted in a marked reduction ofLSO filipin intensity (˜60%). Earlier experiments with geneticrestoration of acid SMase in both CHO and human NPC1 mutant cells (FIGS.22 and 26) showed an approximately 65-70% decrease in LSO filipin.Similarly, addition of rhASM back to human fibroblast at a higher (3μg/mL) concentration (FIG. 27) also resulted in ˜65-70% decrease in LSOfilipin intensity. Thus, the inhibited acid. SMase activity in NPC1cells can be rescued by high-dose exogenous rhASM treatment, whichpresumably overcomes the enzyme inactivation process.

Discussion of Acid SMase Restoration Experiments

The panoply of cellular perturbations triggered by dysfunctionalmutations in individual lysosomal enzymes, co-factors or transportproteins reflects multiple adverse effects of excess substrateaccumulation (Brady 1982; Neufeld 1991). For LSDs involving theaccumulation of certain lipids in LSOs, physical or biochemicalconsequences of accumulation of these excess lipids may lead tosecondary effects on lysosomal/late endosomal processes that, in turn,could amplify the original defect or otherwise contribute to cellularpathology (FIG. 30). In any given LSD, one or more of these secondarydefects may be particularly important, and those involving defects inlysosomal enzymes may be more amenable to correction than thoseinvolving the primary mutation (Neufeld 1980; Brady 2006). Thus,identification of such processes could reveal potentially promisingtherapeutic opportunities for certain LSDs. As an example, severalsphingolipid LSDs acquire a secondary defect in late endosomalcholesterol trafficking which can exacerbate the lipid storage (Puri1999; Lloyd-Evans 2008). In cell culture, correction of this secondarydefect by cellular cholesterol depletion ameliorates a key defect inthese cells, namely, abnormal trafficking of plasma membranesphingolipids to lysosomes instead of the Golgi (Puri 1999). However, itremains unclear how sufficient cellular cholesterol depletion could beachieved to treat patients.

These experiments investigated the converse possibility that restoringSM hydrolysis in LSOs might reduce cholesterol storage and restorenormal cellular membrane traffic. As a test case for this concept, theexperiments studied NPC1 deficient cells. These cells lack amembrane-bound late endosomal protein NPC1, but have a secondary defectin a soluble lysosomal enzyme acid SMase that is amenable toreconstitution. The data show that correction of the secondary acidSMase defect in NPC1-deficient cells markedly reverses the accumulationof two lipids, cholesterol and BMP, and helps restore membrane TfRrecycling—all in the face of a complete absence of the NPC1 protein(FIGS. 24 and 25).

If the suppression of acid SMase activity in NPC1 cells is a secondaryeffect, increasing levels of the enzyme may help overcome theinactivation. In support of this concept, it was observed in the instantexperiments that adding increasing amounts of exogenous rhASM has noeffect on cholesterol levels in WT cells. On the contrary, there is adose-dependent decrease in LSO cholesterol with the addition ofincreasing dose of rhASM to NPC1 cells. The treatment is effective onlyafter the secondary inactivation of acid SMase in these cells isovercome. Once this secondary defect is relieved by excess acid SMase,there is only 25-30% residual cholesterol accumulation, presumablybecause of the primary NPC1 mutation.

In summary, these experiments have shown that key pathological featuresof an LSD cell can be markedly improved by correcting a secondaryabnormality despite complete absence of the protein responsible for theprimary defect. In the case of NPC1 disease, the secondary defect ofacid SMase inactivity is a more feasible therapeutic target than theprimary mutation (Neufeld 1980; Brady 2006). The data in FIGS. 27A-Cdemonstrate this concept at the cell level by showing that the LSOcholesterol trafficking defect in two different human NPC Fbs can bemarkedly corrected by acid SMase enzyme replacement. A reduction of upto 70% of the cholesterol overload in LSOs might translate into clinicalimprovement. Indeed, replacement therapy with acid. SMase is beingtested in humans who have NPA and B disease with primary mutations inthis protein (Schuchman 2007). The challenge with NPC disease is toachieve expression in the brain, but recent advances usingSMPD1-containing adeno-associated virus vectors in mice have shownpromise in this regard (Passini 2005; Dodge 2005).

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While there have been shown and described what are at present consideredthe preferred embodiments of the invention, those skilled in the art maymake various changes and modifications which remain within the scope ofthe invention defined by the appended claims.

1. A composition comprising an active portion comprising apolysaccharide having attached thereto at least one cyclicoligosaccharide, and at least one cell-targeting agent attached to saidactive portion.
 2. The composition of claim 1, wherein said at least onecyclic oligosaccharide is attached as a pendant group from thepolysaccharide.
 3. The composition of claim 1, wherein said cyclicoligosaccharide is a cyclodextrin.
 4. The composition of claim 3,wherein said cyclodextrin is an alkylated cyclodextrin by having atleast one hydroxyl group of the cyclodextrin modified by substitution ofa hydrogen atom of the hydroxyl group with a hydrocarbon group.
 5. Thecomposition of claim 4, wherein said hydrocarbon group contains at leastone and up to six carbon atoms.
 6. The composition of claim 4, whereinsaid alkylated cyclodextrin is a methylated cyclodextrin.
 7. Thecomposition of claim 6, wherein said methylated cyclodextrin is apermethylated cyclodextrin.
 8. The composition of claim 1, wherein saidcyclic oligosaccharide is attached to the polysaccharide via a linker.9. The composition of claim 8, wherein said linker is comprised of atleast one ethyleneoxide moiety.
 10. The composition of claim 1, whereinsaid cell-targeting agent is attached to the polysaccharide.
 11. Thecomposition of claim 1, wherein said cell-targeting agent is attached tothe cyclic oligosaccharide.
 12. The composition of claim 8, wherein thecell-targeting agent is attached to the linker,
 13. The composition ofclaim 1, wherein said cell-targeting agent targets an entity residing ona surface of a cell.
 14. The composition of claim 1, wherein saidcell-targeting agent targets a lysosome.
 15. The composition of claim14, wherein said cell targeting agent is mannose-6-phosphate.
 16. Thecomposition of claim 1, wherein said polysaccharide is a dextran. 17.The composition of claim 1, wherein said polysaccharide has a molecularweight of at least 40,000 Da.
 18. The composition of claim 1, whereinsaid composition further comprises a fluorophore attached to said activeportion.
 19. A pharmaceutical composition comprising the compositionaccording to claim 1, in a pharmaceutically acceptable vehicle.
 20. Amethod for lowering a cholesterol level of a mammal, the methodcomprising administering to said mammal a polymer composition comprisinga polysaccharide having attached thereto at least one cyclicoligosaccharide.
 21. The method of claim 20, wherein said polymercomposition further comprises a cell-targeting agent.
 22. A method fortreating a mammal suffering from a lysosomal storage disorder, themethod comprising administering to said mammal a polymer compositioncomprising a polysaccharide having attached thereto at least one cyclicoligosaccharide.
 23. The method of claim 22, wherein said polymercomposition further comprises a cell-targeting agent.
 24. The method ofclaim 22, wherein said lysosomal storage disorder is a Niemann-Pickdisease.
 25. (canceled)
 26. (canceled)
 27. (canceled)
 28. A method forlowering a cholesterol level of a mammal, the method comprisingadministering to said mammal a sphingomyelinase enzyme.
 29. A method fortreating a mammal suffering from a lysosomal storage disorder, themethod comprising administering to said mammal a sphingomyelinaseenzyme.
 30. The method of claim 29, wherein said lysosomal storagedisorder is a Niemann-Pick disease.